U.S. patent application number 13/733567 was filed with the patent office on 2013-07-04 for assay for identification of lrrk2 inhibitors.
This patent application is currently assigned to MEDICAL RESEARCH COUNCIL. The applicant listed for this patent is MEDICAL RESEARCH COUNCIL. Invention is credited to Dario Alessi, Nicholas Dzamko, R. Jeremy Nichols.
Application Number | 20130171661 13/733567 |
Document ID | / |
Family ID | 44788341 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130171661 |
Kind Code |
A1 |
Dzamko; Nicholas ; et
al. |
July 4, 2013 |
Assay for Identification of LRRK2 Inhibitors
Abstract
A method for assessing the effect of a test compound on LRRK2 in
a cell-based system, the method comprising the steps of a)
assessing the effect of exposing the cell-based system comprising
LRRK2 to the test compound on the phosphorylation state of Ser910
and/or Ser935 of the LRRK2; and/or b) assessing the effect of
exposing the cell-based system comprising LRRK2 to the test
compound on the binding of the LRRK2 to a 14-3-3 polypeptide. The
method may comprise or further comprise the step of assessing the
effect of exposing the cell-based system comprising LRRK2 to the
test compound on the subcellular location of LRRK2. The method is
considered to be useful in assessing the effect of putative LRRK2
inhibitors in cell based systems, including in vivo systems.
Inventors: |
Dzamko; Nicholas; (Dundee,
GB) ; Alessi; Dario; (Dundee, GB) ; Nichols;
R. Jeremy; (Dundee, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDICAL RESEARCH COUNCIL; |
London |
|
GB |
|
|
Assignee: |
MEDICAL RESEARCH COUNCIL
London
GB
|
Family ID: |
44788341 |
Appl. No.: |
13/733567 |
Filed: |
January 3, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12828674 |
Jul 1, 2010 |
8367349 |
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13733567 |
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12763005 |
Apr 19, 2010 |
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12828674 |
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Current U.S.
Class: |
435/7.4 ; 435/23;
530/389.8 |
Current CPC
Class: |
G01N 33/5008 20130101;
C07K 16/40 20130101; G01N 33/573 20130101; G01N 2440/14 20130101;
G01N 2800/2835 20130101; A61P 25/28 20180101; C07K 2317/30
20130101; G01N 2333/912 20130101; C07K 2317/34 20130101; G01N
2500/00 20130101; C12Q 1/485 20130101 |
Class at
Publication: |
435/7.4 ;
530/389.8; 435/23 |
International
Class: |
C12Q 1/48 20060101
C12Q001/48 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 19, 2010 |
GB |
1006502.7 |
Jun 30, 2010 |
GB |
1011010.4 |
Claims
1. An antibody that binds specifically to LRRK2 phosphorylated at
Ser910; or an antibody that binds specifically to LRRK2
phosphorylated at Ser935; or an antibody that binds specifically to
LRRK2 that is not phosphorylated at Ser910; or an antibody that
binds specifically to LRRK2 that is not phosphorylated at
Ser935.
2. A kit of parts comprising two or more of: 1) an antibody that
binds specifically to LRRK2 phosphorylated at Ser910, or an
antibody that binds specifically to LRRK2 that is not
phosphorylated at Ser910; 2) an antibody that binds specifically to
LRRK2 phosphorylated at Ser935, or an antibody that binds
specifically to LRRK2 that is not phosphorylated at Ser935; 3) a
14-3-3 polypeptide, or an antibody that specifically binds to a
14-3-3 polypeptide; and 4) a fluorescently labeled LRRK2
polypeptide, or polynucleotide encoding a fluorescently labeled
LRRK2.
3. A purified preparation or kit of parts comprising an LRRK2
polypeptide or polynucleotide or antibody binding specifically to
LRRK2; and a 14-3-3 polypeptide or polynucleotide or antibody
binding specifically to a 14-3-3 polypeptide.
4. A method of characterising an LRRK2 mutant, the method
comprising the steps of: a) assessing the phosphorylation state of
Ser910 and/or Ser935 of the LRRK2 mutant; and/or b) assessing the
ability of the LRRK2 mutant to bind a 14-3-3 polypeptide.
5. The method of claim 4, wherein the LRRK2 mutant is found in a
patient with Parkinson's disease.
6. The method of claim 4, wherein the method comprises, or further
comprises, the step of assessing the subcellular location of the
LRRK2 mutant, when expressed in a cell-based system.
Description
[0001] The present application is a division of U.S. application
Ser. No. 12/828,674 filed Jul. 1, 2010, which is a
continuation-in-part of U.S. application Ser. No. 12/763,005 filed
Apr. 19, 2010, now abandoned, and claims priority to GB 1011010.4
filed Jun. 30, 2010 and GB1006502.7 filed Apr. 19, 2010. The entire
disclosure of each of these documents are incorporated by reference
herein in their entirety.
[0002] The present invention relates to an assay for assessing
LRRK2 inhibitors.
[0003] Autosomal dominant missense mutations within the gene
encoding for the Leucine Rich Repeat protein Kinase-2 (LRRK2)
predispose humans to Parkinson's disease [1, 2]. Patients with
LRRK2 mutations generally develop Parkinson's disease with clinical
appearance and symptoms indistinguishable from idiopathic
Parkinson's disease at around 60-70 years of age [3]. Mutations in
LRRK2 account for 4% of familial Parkinson's disease, and are
observed in 1% of sporadic Parkinson's disease patients [3].
[0004] LRRK2 is a large enzyme (2527 residues), consisting of
leucine rich repeats (residues 1010-1287), GTPase domain (residues
1335-1504), COR domain (residues 1517-1843), serine/threonine
protein kinase domain (residues 1875-2132) and a WD40 repeat
(residues 2231-2276) [4]. Over 40 missense mutations have been
reported [5]. The activity as well as localisation of a subset of
mutant forms of LRRK2 has been analysed in previous work using
various forms of recombinant LRRK2 expressed and assayed using
diverse approaches [4, 32]. The most frequent mutation comprises an
amino acid substitution of the highly conserved Gly2019 located
within the subdomain VII-DFG motif of the kinase domain to a Ser
residue [5], which enhances the protein kinase activity of LRRK2
around two-fold [6]. This finding suggests that inhibitors of LRRK2
may have utility for the treatment of Parkinson's disease. It was
also reported that various mutants such as LRRK2[R1441C] and
LRRK2[Y1699C] accumulated within discrete cytosolic pools that were
suggested to consist of aggregates of misfolded protein [6].
[0005] The intrinsic protein kinase catalytic activity of LRRK2 is
readily measured in vitro in assays employing peptide substrates
such as LRRKtide [7] or Nictide [8]; see also WO 2008/122789 and
PCT/GB2009/002047. This has made it possible to undertake screens
to identify inhibitors. Recent work has shown that a widely
deployed Rho-kinase (ROCK) inhibitor termed H-1152 also inhibited
LRRK2 with similar potency (IC.sub.50 of 150 nM) [8]. The
multi-target tyrosine kinase inhibitor sunitinib (marketed as
Sutent and also known as SU11248), used for the treatment of renal
cell carcinoma and other cancers, has recently been demonstrated to
inhibit LRRK2 (IC.sub.50 of 20 nM) [8-10]. We have also found that
H-1152 and sunitinib inhibit the LRRK2[G2019S] mutant two to
four-fold more potently than wild type LRRK2 [8]. Based on
molecular modelling of the LRRK2 kinase domain we have designed a
drug resistant LRRK2[Ala2016Thr] mutant that was normally active,
but 32-fold less sensitive to H-1152 and 12-fold less sensitive to
sunitinib [8].
[0006] A bottleneck in the development of LRRK2 inhibitors is how
to assess the relative effectiveness of these compounds in vivo, as
little is known about how LRRK2 is regulated and what its
substrates are. We provide methods that can be used to assess LRRK2
inhibitors in a cell-based system. We demonstrate that LRRK2 kinase
activity regulates phosphorylation of two N-terminal residues
adjacent to the leucine rich repeat domain (Ser910 and Ser935),
which mediate binding to the phospho-adapter 14-3-3 proteins [11].
Consistent with this, H-1152 and sunitinib induced
dephosphorylation of Ser910 and Ser935 thereby disrupting
14-3-3-interaction with wild type LRRK2 and LRRK2[G2019S], but not
with the drug resistant LRRK2[Ala2016Thr] mutant. We provide
evidence that disruption of 14-3-3-binding induces LRRK2 to
accumulate within cytoplasmic pools, similar in appearance to those
reported previously for the LRRK2[R1441C] and LRRK2[Y1699C]
mutants. Phosphorylation of Ser910 and Ser935 or 14-3-3 binding, or
subcellular location of LRRK2, can be used to monitor the efficacy
of LRRK2 inhibitors.
[0007] A first aspect of the invention provides a method for
assessing the effect of a test compound on LRRK2 in a cell-based
system, the method comprising the steps of
a) assessing the effect of exposing the cell-based system
comprising LRRK2 to the test compound on the phosphorylation state
of Ser910 and/or Ser935 of the LRRK2; and/or b) assessing the
effect of exposing the cell-based system comprising LRRK2 to the
test compound on the binding of the LRRK2 to a 14-3-3
polypeptide.
[0008] In some embodiments, the method may comprise, or further
comprise the step of assessing the effect of exposing the
cell-based system comprising LRRK2 to the test compound on the
subcellular location of LRRK2.
[0009] The method may yet further comprise the step of selecting a
compound as being considered to have an inhibitory effect on LRRK2
in a cell-based system, wherein a test compound is so selected if
the phosphorylation of Ser910 and/or Ser935 of the LRRK2 is reduced
following the exposure; and/or the binding of the LRRK2 to a 14-3-3
polypeptide is reduced following the exposure.
[0010] The test compound may typically be a compound that has
already been selected as a possible inhibitor of LRRK2, for example
using an in vitro assay, for example an assay using LRRKtide or
Nictide as an LRRK2 substrate polypeptide. Examples of assays
suitable for selecting a compound as a possible inhibitor of LRRK2
are described in, for example, WO 2008/122789 and
PCT/GB2009/002047.
[0011] Typically phosphorylation of Ser910 is assessed using an
antibody that binds specifically to LRRK2 phosphorylated at Ser910
or an antibody that binds specifically to LRRK2 that is not
phosphorylated at Ser910.
[0012] Typically phosphorylation of Ser935 is assessed using an
antibody that binds specifically to LRRK2 phosphorylated at Ser935
or an antibody that binds specifically to LRRK2 that is not
phosphorylated at Ser935.
[0013] By an antibody that binds specifically to LRRK2
phosphorylated at Ser910 is meant an antibody that binds to LRRK2
phosphorylated at Ser910, but not to LRRK2 that is not
phosphorylated at Ser910, or to other phosphorylated serine
residues. Similarly an antibody that binds specifically to LRRK2
phosphorylated at Ser935 does not bind to LRRK2 that is not
phosphorylated at Ser935, or to other phosphorylated serine
residues. An antibody that binds generally to phosphorylated serine
residues is not an antibody that binds specifically to LRRK2
phosphorylated at Ser910 or an antibody that binds specifically to
LRRK2 phosphorylated at Ser935.
[0014] Similar considerations apply in relation to an antibody that
binds specifically to LRRK2 that is not phosphorylated at Ser910 or
an antibody that binds specifically to LRRK2 that is not
phosphorylated at Ser935. An antibody that binds specifically to
LRRK2 that is not phosphorylated at Ser910 does not bind to LRRK2
that is phosphorylated at Ser910. An antibody that binds
specifically to LRRK2 that is not phosphorylated at Ser935 does not
bind to LRRK2 that is phosphorylated at Ser935.
[0015] Methods of generating and using such antibodies will be
apparent to those skilled in the art. Examples of such antibodies
and methods of generating and using them are described in the
Examples. The antibodies may be polyclonal or monoclonal.
[0016] As an example an ELISA type assay may be particularly
useful, as will be well known to those skilled in the art.
[0017] Binding of the LRRK2 to a 14-3-3 polypeptide may be assessed
by any suitable technique for assessing protein:protein
interaction. Typically a FRET (fluorescence resonance energy
transfer) technique may be used, as discussed further below. Other
techniques that may be useful may make use of immunoprecipitation
techniques. For example, immunoprecipitation may be with an
antibody that binds specifically to LRRK2; or may be with an
antibody that binds specifically to a 14-3-3 polypeptide, as will
be apparent to the skilled person. Antibodies that bind
specifically to a 14-3-3 polypeptide will be well known to those
skilled in the art and are commercially available. Alternatively,
immunoprecipitation may be with an antibody that binds specifically
to a tag present on recombinant LRRK2; or with an antibody that
binds specifically to a tag present on recombinant 14-3-3
polypeptide, as will also be apparent to the skilled person.
[0018] As an example, it is considered that detection of
phospho/dephospho-LRRK coupled with either 14-3-3 co-pull down or
an anti-LRRK2 antibody (not phosphorylation state dependent) can be
carried out using Invitrogen's Alpha-Elisa technologies, which
would be useful in achieving a high throughput screening system.
Multiplex assays using Luminex beads or plate based
electrochemiluminescence (MSD; meso scale discovery) detection
could also be used.
[0019] Details of Alpha screen technology (Perkin Elmer) applicable
to both protein:protein and phosphoprotein detection (Sure fire
kits developed and sold for MAPK, JAK/STAT and AKT pathways) can be
found at, for example,
http://las.perkinelmer.co.uk/Catalog/CategoryPage.htm?CategoryID=AlphaTec-
h&M=BIO
[0020] Details of Luminex technology applicable to phospho protein
detection and protein:protein and total protein quantitation can be
found at, for example,
http://www.luminexcorp.com/applications/cellular_signaling.html
[0021] An example of the use of such technology is described in
reference Khan I H, Zhao J., Ghosh, P. Ziman, M., Sweeney C, Kung H
J and Luciw P A (2010) Assay Drug Dev technology 8, 27-36.
[0022] In MSD technology the principles of capture onto surface of
plate and antibody detection are the same as any ELISA but the mode
of detection uses electrochemiluminescence via Ruthenium tagged
probes, and the technology allows multiplexing in the well through
an array format.
http://www.mesoscale.com/CatalogSystemWeb/WebRoot/literature/brochures/pd-
f/techBrochure.pdf
[0023] Quantitative Stable Isotope Labelling with Amino acids in
Cell culture (SILAC)-based mass spectrometry may be used to
identify and quantitate proteins associated with immunoprecipitates
of LRRK2 (or of a 14-3-3 polypeptide). Other immunoprecipitate
methods may be used, as will be well known to those skilled in the
art. For example digoxygenin labeled 14-3-3 polypeptide may be
used. Some examples of such methods are described in the Examples.
As noted above, other techniques for assessing protein:protein
interactions in cells or cell extracts may also be used. For
example, a fluorescence resonance energy transfer (FRET) based
system may be used if the interaction of a recombinant LRRK2 and
recombinant 14-3-3 polypeptide is being assessed, for example if
both LRRK2 and 14-3-3 polypeptide are both tagged with a
fluorescent polypeptide.
[0024] Thus, the molecular interaction between LRRK2 and 14-3-3
proteins (and the effects of test compounds) could be investigated
using a FRET-based method such as FLIM-FRET on a microscope such as
a multiphoton microscope. As an example, a construct for expressing
Chemy-tagged wild type 14-3-3 isoform or (as a control) an inactive
mutant of Chemy-tagged 14-3-3 isoform such as 14-3-3 zeta [E180K]
that does not bind phospho targets may be transfected into a cell
line stably expressing wild type GFP-LRRK2 or (as controls)
GFP-LRRK2[S910A/S935A]. FRET (fluorescence resonance energy
transfer) can occur when the GFP and mCherry fluorophores are
brought together by virtue of the binding of LRRK2 to 14-3-3 which
will in turn affect their fluorescence lifetime, which can be
detected. Using FLIM (fluorescence lifetime imaging microscopy) we
can generate a spatial distribution of the cell where sites of
strong protein-protein interaction (and therefore FRET) and weak
interaction or no interaction can be recognised (by colour coding:
see, for example, Lieres et al. 2009 Quantitative analysis of
chromatin compaction in living cells using FLIM-FRET. J Cell Biol.
2009 Nov. 16; 187(4):481-96.). No FLIM-FRET should be observed
between GFP-LRRK2[S910A/S935A] and mCherry-14-3-3 or between wild
type GFP-LRRK2 and inactive 14-3-3 polypeptide.
[0025] Commonly used FRET pairs include CFP (donor) and YFP
(acceptor) as well as GFP (donor) and Chemy (acceptor). In cases
where the donor and acceptor fluorophores are both excited with the
same excitation light wavelength, e.g. in case of the FRET pair
GFP-YFP, a special kind of FRET termed enhanced acceptor
fluorescence (EAF) can be detected. Examples of further references
concerning FRET techniques include Wallrabe & Periasamy (2005)
Current Opinion in Biotechnology Volume 16, Issue 1, February 2005,
Pages 19-27; Imaging protein molecules using FRET and FLIM
microscopy; Ai et al (2008) Nature Methods 5, 401-403 Fluorescent
protein FRET pairs for ratiometric imaging of dual biosensors;
Shaner et al. (2004) Nat Biotechnol 22: 1567-1572 Improved
monomeric red, orange and yellow fluorescent proteins derived from
Discosoma sp. red fluorescent protein.
[0026] Assessing the effect of exposing the cell-based system
comprising LRRK2 to the test compound on the phosphorylation state
of Ser910 and/or Ser935 of the LRRK2; and/or assessing the effect
of exposing the cell-based system comprising LRRK2 to the test
compound on the binding of the LRRK2 to a 14-3-3 polypeptide may be
done by (or further assessed by) assessing the subcellular
localisation of LRRK2. It is considered that reducing the
phosphorylation of Ser910 and/or Ser935 and/or reducing the binding
of LRRK2 to a 14-3-3 polypeptide increases the amount of LRRK2
polypeptide present in cytoplasmic pools (as opposed to being
diffusely located throughout the cytoplasm). The subcellular
location of LRRK2 may be assessed using techniques well known to
those skilled in the art, for example using immunohistochemistry or
fluorescence microscopy, for example using a recombinant LRRK2
polypeptide with a fluorescent protein (for example GFP) tag.
Examples of such techniques are given in the Examples.
[0027] In an embodiment, the method of the invention may comprise
or further comprise the step of assessing the effect of exposing
the cell-based system comprising LRRK2 to the test compound on the
subcellular location of LRRK2.
[0028] The cell based system may be an in vitro cell system. For
example, the assay may be performed on cell lines. Examples of
suitable cell lines are considered to include Swiss 3T3 cells or
HEK-293 cells. Other suitable cells include, for example, EBV
transformed lymphoblastoid cells derived from a human subject
expressing wild-type LRRK2, or from a human subject homozygous for
LRRK2[G2019S] (or other LRRK2 mutant associated with Parkinsonism).
A neuronal cell line may also be used. Suitable cell lines may also
be cell lines that express a recombinant LRRK2 and/or recombinant
14-3-3 polypeptide. Suitable cells for such expression are
considered to include T-Rex cells, as described in the Examples.
The cells, for example T-Rex cells may express the recombinant
LRRK2 or recombinant 14-3-3 polypeptide in an inducible manner, as
will be well known to those skilled in the art. For example, cells
may be induced to express the desired recombinant polypeptide by
inclusion of doxycycline in the culture medium, for example as
described in the Examples.
[0029] The 14-3-3 polypeptide may typically be or comprise the
human beta, eta, theta, zeta, gamma or epsilon isoform. It is
preferred that the 14-3-3 polypeptide is not solely the human sigma
isoform. Examples of 14-3-3 polypeptide sequences are shown below.
The skilled person will readily be able to identify other 14-3-3
polypeptide sequences from databases. For example, the Homologene
feature of the NCBI database may be used.
TABLE-US-00001 Human 14-3-3 beta SEQ ID NO: 1
MTMDKSELVQKAKLAEQAERYDDMAAAMKAVTEQGHELSNEERNLLSVAYKNVVGARRSSWR
VISSIEQKTERNEKKQQMGKEYREKIEAELQDICNDVLELLDKYLIPNATQPESKVFYLKMK
GDYFRYLSEVASGDNKQTTVSNSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYEILNS
PEKACSLAKTAFDEAIAELDTLNEESYKDSTLIMQLLRDNLTLWTSENQGDEGDAGEGEN Mouse
14-3-3 beta SEQ ID NO: 2
MTMDKSELVQKAKLAEQAERYDDMAAAMKAVTEQGHELSNEERNLLSVAYKNVVGARRSSWR
VISSIEQKTERNEKKQQMGKEYREKIEAELQDICNDVLELLDKYLILNATQAESKVFYLKMK
GDYFRYLSEVASGENKQTTVSNSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYEILNS
PEKACSLAKTAFDEAIAELDTLNEESYKDSTLIMQLLRDNLTLWTSENQGDEGDAGEGEN Human
14-3-3 epsilon SEQ ID NO: 3
MDDREDLVYQAKLAEQAERYDEMVESMKKVAGMDVELTVEERNLLSVAYKNVIGARRASWRI
ISSIEQKEENKGGEDKLKMIREYRQMVETELKLICCDILDVLDKHLIPAANTGESKVFYYKM
KGDYHRYLAEFATGNDRKEAAENSLVAYKAASDIAMTELPPTHPIRLGLALNFSVFYYEILN
SPDRACRLAKAAFDDAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDMQGDGEEQNKEALQ
DVEDENQ Mouse 14-3-3 epsilon SEQ ID NO: 4
MDDREDLVYQAKLAEQAERYDEMVESMKKVAGMDVELTVEERNLLSVAYKNVIGARRASWRI
ISSIEQKEENKGGEDKLKMIREYRQMVETELKLICCDILDVQDKHLIPAANTGESKVFYYKM
KGDYHRYLAEFATGNDRKEAAENSLVAYKAASDIAMTELPPTHPIRLGLALNFSVFYYEILN
SPDRACRLAKAAFDDAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDMQGDGEEQNKEALQ
DVEDENQ Human 14-3-3 eta SEQ ID NO: 5
MGDREQLLQRARLAEQAERYDDMASAMKAVTELNEPLSNEDRNLLSVAYKNVVGARRSSWRV
ISSIEQKTMADGNEKKLEKVKAYREKIEKELETVCNDVLSLLDKFLIKNCNDFQYESKVFYL
KMKGDYYRYLAEVASGEKKNSVVEASEAAYKEAFEISKEQMQPTHPIRLGLALNFSVFYYEI
QNAPEQACLLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDEEAGEGN Mouse
14-3-3 eta SEQ ID NO: 6
MGDREQLLQRARLAEQAERYDDMASAMKAVTELNEPLSNEDRNLLSVAYKNVVGARRSSWRV
ISSIEQKTMADGNEKKLEKVKAYREKIEKELETVCNDVLALLDKFLIKNCNDFQYESKVFYL
KMKGDYYRYLAEVASGEKKNSVVEASEAAYKEAFEISKEHMQPTHPIRLGLALNFSVFYYEI
QNAPEQACLLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDEEAGEGN Human
14-3-3 gamma SEQ ID NO: 7
MVDREQLVQKARLAEQAERYDDMAAAMKNVTELNEPLSNEERNLLSVAYKNVVGARRSSWRV
ISSIEQKTSADGNEKKIEMVRAYREKIEKELEAVCQDVLSLLDNYLIKNCSETQYESKVFYL
KMKGDYYRYLAEVATGEKRATVVESSEKAYSEAHEISKEHMQPTHPIRLGLALNYSVFYYEI
QNAPEQACHLAKTAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDDDGGEGNN Mouse
14-3-3gamma SEQ ID NO: 8
MVDREQLVQKARLAEQAERYDDMAAAMKNVTELNEPLSNEERNLLSVAYKNVVGARRSSWRV
ISSIEQKTSADGNEKKIEMVRAYREKIEKELEAVCQDVLSLLDNYLIKNCSETQYESKVFYL
KMKGDYYRYLAEVATGEKRATVVESSEKAYSEAHEISKEHMQPTHPIRLGLALNYSVFYYEI
QNAPEQACHLAKTAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDDDGGEGNN Human
14-3-3 theta SEQ ID NO: 9
MEKTELIQKAKLAEQAERYDDMATCMKAVTEQGAELSNEERNLLSVAYKNVVGGRRSAWRVI
SSIEQKTDTSDKKLQLIKDYREKVESELRSICTTVLELLDKYLIANATNPESKVFYLKMKGD
YFRYLAEVACGDDRKQTIDNSQGAYQEAFDISKKEMQPTHPIRLGLALNFSVFYYEILNNPE
LACTLAKTAFDEAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDSAGEECDAAEGAEN Mouse
14-3-3 theta SEQ ID NO: 10
MEKTELIQKAKLAEQAERYDDMATCMKAVTEQGAELSNEERNLLSVAYKNVVGGRRSAWRVI
SSIEQKTDTSDKKLQLIKDYREKVESELRSICTTVLELLDKYLIANATNPESKVFYLKMKGD
YFRYLAEVACGDDRKQTIENSQGAYQEAFDISKKEMQPTHPIRLGLALNFSVFYYEILNNPE
LACTLAKTAFDEAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDSAGEECDAAEGAEN Human
14-3-3 zeta SEQ ID NO: 11
MDKNELVQKAKLAEQAERYDDMAACMKSVTEQGAELSNEERNLLSVAYKNVVGARRSSWRVV
SSIEQKTEGAEKKQQMAREYREKIETELRDICNDVLSLLEKFLIPNASQAESKVFYLKMKGD
YYRYLAEVAAGDDKKGIVDQSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYEILNSPE
KACSLAKTAFDEAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDTQGDEAEAGEGGEN Mouse
14-3-3 zeta SEQ ID NO: 12
MDKNELVQKAKLAEQAERYDDMAACMKSVTEQGAELSNEERNLLSVAYKNVVGARRSSWRVV
SSIEQKTEGAEKKQQMAREYREKIETELRDICNDVLSLLEKFLIPNASQPESKVFYLKMKGD
YYRYLAEVAAGDDKKGIVDQSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYEILNSPE
KACSLAKTAFDEAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDTQGDEAEAGEGGEN Human
14-3-3 sigma SEQ ID NO: 13
MERASLIQKAKLAEQAERYEDMAAFMKGAVEKGEELSCEERNLLSVAYKNVVGGQRAAWRVL
SSIEQKSNEEGSEEKGPEVREYREKVETELQGVCDTVLGLLDSHLIKEAGDAESRVFYLKMK
GDYYRYLAEVATGDDKKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFHYEIANS
PEEAISLAKTTFDEAMADLHTLSEDSYKDSTLIMQLLRDNLTLWTADNAGEEGGEAPQEPQS
Mouse 14-3-3 sigma SEQ ID NO: 14
MERASLIQKAKLAEQAERYEDMAAFMKSAVEKGEELSCEERNLLSVAYKNVVGGQRAAWRVL
SSIEQKSNEEGSEEKGPEVKEYREKVETELRGVCDTVLGLLDSHLIKGAGDAESRVFYLKMK
GDYYRYLAEVATGDDKKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFHYEIANS
PEEAISLAKTTFDEAMADLHTLSEDSYKDSTLIMQLLRDNLTLWTADSAGEEGGEAPEEPQS
[0030] The 14-3-3 polypeptide may comprise a tag sequence, as will
be well known to those skilled in the art. For example, a tag
useful in a FRET system may be used. For example a fluorescent
protein tag, for example a Chemy tag may be used. It is considered
that the 14-3-3 polypeptide may be in the form of a dimer
(typically a homodimer) when bound to the LRRK2 polypeptide, as is
generally considered to be the case for binding of 14-3-3
polypeptide to a phosphorylated polypeptide. Typically the 14-3-3
polypeptide is a full length 14-3-3 polypeptide.
[0031] The recombinant LRRK2 may be an LRRK2 that is tagged, for
example with a fluorescent polypeptide moiety, for example a GST
moiety or Green Fluorescent Protein (GFP) moiety or a FLAG moiety,
for example as described in the Examples. The LRRK2 polypeptide may
be wild-type LRRK2 or may be an LRRK2 mutant, for example
LRRK2[G2019S]. Typically the LRRK2 does not have the drug-resistant
A2016T mutation. Typically the LRRK2 is not a kinase inactive
mutant. Typically the LRRK2 has Serine residues at positions 910
and 935 (numbering of full length wild type LRRK2). Typically the
sequences surrounding these serine residues are also unchanged from
wild-type LRKK2. In particular, residues identified in FIG. 3G
typically are retained i.e. basic residues -3 and -4 positions, Ser
residue at the -2 position, Asn at the -1 position and a large
hydrophobic residue at the +1 position. Typically the LRRK2 is full
length LRRK2.
[0032] Control cells in which the LRRK2 has the drug-resistant
A2016T mutation may be useful. Control cells in which the LRRK2 has
a mutation (for example to Alanine) at one or both of positions 910
and 935 (numbering of full length wild type LRRK2) may be useful.
Control cells in which the LRRK2 is a kinase inactive mutant may
also be useful.
[0033] Cell lines stably expressing FLAG or GST tagged LRRK2 may be
particularly useful. Cell lines expressing LRRK2 and a 14-3-3
polypeptide tagged with fluorescent tags compatible for performing
FRET may be useful. Examples of FRET donor-acceptor pairs will be
well known to those skilled in the art and some examples are given
above. For example the LRRK2 may be tagged with a GFP moiety whilst
the 14-3-3 polypeptide may be tagged with a Chemy moiety.
[0034] Neuronal cell lines or blood cell lines may also be
particularly useful. Any cell line where LRRK2 is endogenously
expressed may also be useful.
[0035] The LRRK2 is typically human LRRK2, but may alternatively be
another mammalian LRRK2, for example LRRK2 of a laboratory animal
or of a tissue or organ assay system considered useful in assessing
a potential inhibitor of LRRK2. Thus, the LRRK2 may be a laboratory
rodent LRRK2 (for example mouse, rabbit or rat) or may be a
laboratory primate LRRK2, for example a monkey LRRK2. An assay of
the present invention may, for example, be useful in assessing the
effect of a test compound on LRRK2 in brain tissue of a laboratory
animal, for example a mouse or a monkey.
[0036] The LRRK2 polypeptide can be human LRRK2 having a naturally
occurring mutation of wild type human LRRK2; or a fusion thereof.
The naturally occurring mutation of human LRRK2 may be a mutation
associated with Parkinson's Disease (PD). As noted above, the
mutation, using the numbering of wild type human LRRK2, may be
G2019S. This mutation is considered to enhance the protein kinase
activity of LRRK2, as discussed further in Jaleel et al (2007)
supra or in PCT/GB2008/001211, supra.
[0037] The mutation, using the numbering of wild type human LRRK2,
may alternatively be R1441c, R1441G, Y1699C, R1914H, 12012T,
12020T, or G2385R. LRRK2 with mutations R1441c, R1441G, Y1699C or
T23561 is considered to have similar protein kinase activity to
wild-type LRRK2. LRRK2 with mutation R1914H or 12012T is considered
to be nearly inactive. LRRK2 with mutation R1441c or Y1699C is
considered to accumulate in cytoplasmic pools (rather than being
diffusely present throughout the cytoplasm) to a greater extent
than wild-type LRRK2. LRRK2 with mutation 12020T is considered to
have activity intermediate between wild-type LRRK2 and LRRK2 with
mutation R1914H or 12012T. LRRK2 with mutation G2385R is also
considered to be nearly inactive. The activities of further mutants
are shown in FIG. 17 of PCT/GB2008/001211, supra.
[0038] It may be helpful to test compounds against more than one
LRRK2 polypeptide; for example against more than one mutant LRRK2
polypeptide. This may assist in deciding on further compounds to
design and test.
[0039] It is particularly preferred, although not essential, that
the LRRK2 polypeptide has at least 30% of the enzyme activity of
full-length human LRRK2 with respect to the phosphorylation of
full-length human moesin on residue Thr558 or Thr526; or the
phosphorylation of a peptide substrate encompassing such a residue
(for example RLGRDKYKTLRQIRQ (SEQ ID NO:15) or
RLGRDKYKTLRQIRQGNTKQR (SEQ ID NO:16) or RLGWWRFYTLRRARQGNTKQR (SEQ
ID NO:17). It is more preferred if the LRRK2 polypeptide has at
least 50%, preferably at least 70% and more preferably at least 90%
of the enzyme activity of full-length human LRRK2 with respect to
the phosphorylation of full-length human moesin on residue Thr558
or Thr526; or the phosphorylation of a peptide substrate
encompassing such a residue, as discussed above; or of
RLGWWRFYTLRRARQGNTKQR.
[0040] Accession numbers for mammalian LRRK2 sequences in the NCBI
database include:
AAV63975.1 human XP.sub.--001168494.1 Pan troglodytes, (chimpanzee)
XP.sub.--615760.3 Bos Taurus (domestic cow) XP.sub.--543734.2 Canis
familiaris (dog) NP.sub.--080006.2 Mus musculus (mouse)
XP.sub.--235581.4 Rattus norvegicus (rat)
[0041] Numerous further examples of mammalian and non-mammalian
LRRK2 polypeptide sequences can be accessed in the sequence
databases accessible from the NCBI Medline.TM. service, as will be
well known to the person skilled in the art.
[0042] By "variants" of a polypeptide we include insertions,
deletions and substitutions, either conservative or
non-conservative. In particular we include variants of the
polypeptide where such changes do not substantially alter the
protein kinase activity or ability to be phosphorylated, or the
interaction between LRRK2 and 14-3-3 polypeptide, as appropriate.
The skilled person will readily be able to design and test
appropriate variants, based on, for example, comparison of
sequences of examples of each polypeptide, for example from
different species. The skilled person will readily be able to
determine where insertions or deletions can be made; or which
residues can appropriately be left unchanged; replaced by a
conservative substitution; or replaced by a non-conservative
substitution. The variant polypeptides can readily be tested, for
example as described in the Examples.
[0043] By "conservative substitutions" is intended combinations
such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys,
Arg; and Phe, Tyr.
[0044] The three-letter or one letter amino acid code of the
IUPAC-IUB Biochemical Nomenclature Commission is used herein, with
the exception of the symbol Zaa, defined above. In particular, Xaa
represents any amino acid. It is preferred that at least the amino
acids corresponding to the consensus sequences defined herein are
L-amino acids.
[0045] It is particularly preferred if the polypeptide variant has
an amino acid sequence which has at least 65% identity with the
amino acid sequence of the relevant human polypeptide, more
preferably at least 70%, 71%, 72%, 73% or 74%, still more
preferably at least 75%, yet still more preferably at least 80%, in
further preference at least 85%, in still further preference at
least 90% and most preferably at least 95% or 97% identity with the
amino acid sequence of the relevant human polypeptide.
[0046] It is still further preferred if a protein kinase variant
has an amino acid sequence which has at least 65% identity with the
amino acid sequence of the catalytic domain of the human
polypeptide, more preferably at least 70%, 71%, 72%, 73% or 74%,
still more preferably at least 75%, yet still more preferably at
least 80%, in further preference at least 83 or 85%, in still
further preference at least 90% and most preferably at least 95% or
97% identity with the relevant human amino acid sequence.
[0047] It will be appreciated that the catalytic domain of a
protein kinase-related polypeptide may be readily identified by a
person skilled in the art, for example using sequence comparisons
as described below. Protein kinases show a conserved catalytic
core, as reviewed in Johnson et al (1996) Cell, 85, 149-158 and
Taylor & Radzio-Andzelm (1994) Structure 2, 345-355. This core
folds into a small N-terminal lobe largely comprising anti-parallel
.beta.-sheet, and a large C-terminal lobe which is mostly
.alpha.-helical.
[0048] The percent sequence identity between two polypeptides may
be determined using suitable computer programs, for example the GAP
program of the University of Wisconsin Genetic Computing Group and
it will be appreciated that percent identity is calculated in
relation to polypeptides whose sequence has been aligned
optimally.
[0049] The alignment may alternatively be carried out using the
Clustal W program (Thompson et al., 1994). The parameters used may
be as follows:
Fast pairwise alignment parameters: K-tuple (word) size; 1, window
size; 5, gap penalty; 3, number of top diagonals; 5. Scoring
method: x percent. Multiple alignment parameters: gap open penalty;
10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.
[0050] The alignment may alternatively be carried out using the
program T-Coffee, or EMBOSS.
[0051] The residue corresponding (equivalent) to, for example,
Ser910 of full-length human LRRK2 may be identified by alignment of
the sequence of the polypeptide with that of full-length human
LRRK2 in such a way as to maximise the match between the sequences.
The alignment may be carried out by visual inspection and/or by the
use of suitable computer programs, for example the GAP program of
the University of Wisconsin Genetic Computing Group, which will
also allow the percent identity of the polypeptides to be
calculated. The Align program (Pearson (1994) in: Methods in
Molecular Biology, Computer Analysis of Sequence Data, Part II
(Griffin, A M and Griffin, H G eds) pp 365-389, Humana Press,
Clifton). Thus, residues identified in this manner are also
"corresponding residues".
[0052] It will be appreciated that in the case of truncated forms
of (for example) LRRK2 or in forms where simple replacements of
amino acids have occurred it is facile to identify the
"corresponding residue".
[0053] It is preferred that the polypeptides used in the screen are
mammalian, preferably human (or a species useful in agriculture or
as a domesticated or companion animal, for example dog, cat, horse,
cow), including naturally occurring allelic variants (including
splice variants). The polypeptides used in the screen may comprise
a GST portion or may be biotinylated or otherwise tagged, for
example with a 6His, HA, myc or other epitope tag, as known to
those skilled in the art, or as mentioned above or as described in
the Examples. This may be useful in purifying and/or detecting the
polypeptide(s).
[0054] The effect of the compound may be determined by comparing
the phosphorylation of residues Ser910 or Ser935, or the binding of
14-3-3 polypeptide, or the subcellular localization of LRRK2 in the
presence of different concentrations of the compound, for example
in the absence and in the presence of the compound, for example at
a concentration of about 100 .mu.M, 30 .mu.M, 10 .mu.M, 3 .mu.M, 1
.mu.M, 0.1 .mu.M, 0.01 .mu.M and/or 0.001 .mu.M.
[0055] It may be useful to compare the effect of the test compound
with the effect of compounds considered to be inhibitors of LRRK2,
for example H-1152 and/or sunitinib.
[0056] The cell based system may be an ex vivo cell system. The
cells may be in the form of a sample of tissue or an organ. The
sample may be a sample of blood, kidney, brain or spleen (or other
tissue in which LRRK2 is highly expressed).
[0057] The cell based system may be an in vivo system. For example
the cell-based system comprising LRRK2 may have been exposed to the
test compound in a test animal. Suitable ways of exposing a test
animal to the test compound will be well known to those skilled in
the art. Typically the compound may be formulated for
administration by injection or for oral administration but other
administration routes may be used, as will be apparent to the
skilled person. A sample for analysis may be obtained from the test
animal by invasive, minimally invasive or non-invasive techniques.
For example, a blood sample (minimally invasive) may be analysed;
or a sample of brain tissue (invasive), which may require sacrifice
of the animal.
[0058] The assessing of the phosphorylation state of Ser910 and/or
Ser935 of the LRRK2; and/or the assessing of the binding of the
LRRK2 to a 14-3-3 polypeptide and/or the assessing of the
subcellular location of LRRK2 may be performed on cells obtained
from the test animal. For example, the cells obtained from the test
animal may be cells obtained in blood from the test animal.
[0059] The cell based system may be a lymphoblastoid cell-based
system. Lymphoblastoid cells may be present in a blood sample from
a test animal. A macrophage cell line (for example RAW cell line)
system may be useful. A system making use of macrophages obtained
from blood from human volunteers may also be useful.
[0060] The method is considered to be useful in identifying
compounds that modulate, for example inhibit, the protein kinase
activity of LRRK2 (or the phosphorylation of Ser910 and/or Ser935
or interaction between LRRK2 and a 14-3-3 polypeptide or
accumulation of LRRK2 in cytoplasmic pools) in a cell-based system.
A compound that modulates, for example inhibits, the protein kinase
activity of LRRK2 (or the phosphorylation of Ser910 and/or Ser935
or interaction between LRRK2 and a 14-3-3 polypeptide or
accumulation of LRRK2 in cytoplasmic pools) in a cell-based system
may be useful in the treatment of Parkinson's Disease (for example
idiopathic Parkinson's Disease or late-onset Parkinson's Disease)
or Parkinson ism.
[0061] A compound that modulates, for example inhibits, the protein
kinase activity of LRRK2 (or the phosphorylation of Ser910 and/or
Ser935 or interaction between LRRK2 and a 14-3-3 polypeptide or
accumulation of LRRK2 in cytoplasmic pools) in a cell-based system,
may also be useful in other neurodegenerative conditions.
[0062] The compound may be one which binds to or near a region of
contact between a LRRK2 polypeptide and a substrate polypeptide, or
may be one which binds to another region and, for example, induces
a conformational or allosteric change which stabilises (or
destabilises) the complex; or promotes (or inhibits) its formation.
The compound may bind to the LRRK2 polypeptide or to the substrate
polypeptide so as to increase the LRRK2 polypeptide protein kinase
activity by an allosteric effect. This allosteric effect may be an
allosteric effect that is involved in the natural regulation of the
LRRK2 polypeptide's activity.
[0063] The compounds identified in the methods may themselves be
useful as a drug or they may represent lead compounds for the
design and synthesis of more efficacious compounds.
[0064] The compound may be a drug-like compound or lead compound
for the development of a drug-like compound for each of the above
methods of identifying a compound. It will be appreciated that the
said methods may be useful as screening assays in the development
of pharmaceutical compounds or drugs, as well known to those
skilled in the art.
[0065] The term "drug-like compound" is well known to those skilled
in the art, and may include the meaning of a compound that has
characteristics that may make it suitable for use in medicine, for
example as the active ingredient in a medicament. Thus, for
example, a drug-like compound may be a molecule that may be
synthesised by the techniques of organic chemistry, less preferably
by techniques of molecular biology or biochemistry, and is
preferably a small molecule, which may be of less than 5000
daltons. A drug-like compound may additionally exhibit features of
selective interaction with a particular protein or proteins and be
bioavailable and/or able to penetrate cellular membranes, but it
will be appreciated that these features are not essential.
[0066] The term "lead compound" is similarly well known to those
skilled in the art, and may include the meaning that the compound,
whilst not itself suitable for use as a drug (for example because
it is only weakly potent against its intended target, non-selective
in its action, unstable, difficult to synthesise or has poor
bioavailability) may provide a starting-point for the design of
other compounds that may have more desirable characteristics.
[0067] It will be understood that it will be desirable to identify
compounds that may modulate the activity of the protein kinase in
vivo. Thus it will be understood that reagents and conditions used
in the method may be chosen such that the interactions between, for
example, the LRRK2 polypeptide and a substrate polypeptide, are
substantially the same as between the human LRRK2 and an endogenous
human substrate polypeptide. Typically a method of the invention
may be performed in a human cell-based system, optionally
expressing human recombinant polypeptides. It will be appreciated
that the compound may bind to the LRRK2 polypeptide, or may bind to
the substrate polypeptide.
[0068] The compounds that are tested in the screening methods of
the invention or in other assays in which the ability of a compound
to modulate the protein kinase activity of an LRRK2 polypeptide,
may be measured, may be (but do not have to be) compounds that have
been selected and/or designed (including modified) using molecular
modelling techniques, for example using computer techniques. The
selected or designed compound may be synthesised (if not already
synthesised) and tested for its effect on the LRRK2 polypeptide,
for example its effect on the protein kinase activity. The compound
may be tested in a screening method of the invention.
[0069] The compounds that are tested may be compounds that are
already considered likely to be able to modulate the activity of a
protein kinase; or may be compounds that have not been selected on
the basis of being likely to modulate the activity of a protein
kinase. Thus, the compounds tested may be compounds forming at
least part of a general, unselected compound bank; or may
alternatively be compounds forming at least part of a pre-selected
compound bank, for example a bank of compounds pre-selected on the
basis of being considered likely to modulate the activity of a
protein kinase.
[0070] It will be appreciated that screening assays which are
capable of high throughput operation will be particularly
preferred.
[0071] As will be apparent to those skilled in the art, it may be
desirable to assess what effect the compound has on other protein
kinases. For example, it may be desirable to assess the effect of
the compound on phosphorylation of substrates of other protein
kinases, for example substrates of RockII, in order to distinguish
between LRRK2 and ROCK inhibitors. For example, as shown in, for
example, FIGS. 20 and 22 of PCT/GB2008/001211, supra or discussed
in the legends thereto, the substrate preferences of LRRK2 and
Rock-II are different. As an example, LRRK2 does not phosphorylate
MYPT, while RockII does phosphorylate MYPT.
[0072] Information on PD models, biomarkers and assessment
techniques, in/against which it may be appropriate further to test
compounds identified using the screening methods described herein,
can be found at, for example, the following links, which are
representative of information available to those skilled in the
art.
http://www.ninds.nih.gov/about_ninds/plans/nihparkinsons_agenda.htm#Model-
s http://www.sciencedaily.com/releases/2006/07/060729134653.htm
(mouse model with mitochondrial disturbance)
http://www.sciencedaily.com/releases/2004/10/041005074846.htm
(embryonic stem cell model)
http://en.wikipedia.org/wiki/Parkinson's_disease
[0073] PD animal models include the 6-hydroxydopamine treated
rodent and the MPTP treated primate. Both are based on toxic
destruction of dopaminergic brain cells (and some other types), and
usually employ young, otherwise healthy animals. Because these
models reproduce some key features of Parkinson's disease, they are
considered useful to test emerging new therapies.
[0074] Compounds may also be subjected to other tests, for example
toxicology or metabolism tests, as is well known to those skilled
in the art.
[0075] The screening method of the invention may comprise the step
of synthesising, purifying and/or formulating the selected
compound. The compound may be formulated for pharmaceutical use,
for example for use in in vivo trials in animals or humans.
[0076] A further aspect of the invention provides an antibody that
binds specifically to LRRK2 phosphorylated at Ser910; or an
antibody that binds specifically to LRRK2 phosphorylated at Ser935;
or an antibody that binds specifically to LRRK2 that is not
phosphorylated at Ser910; or an antibody that binds specifically to
LRRK2 that is not phosphorylated at Ser935.
[0077] A further aspect of the invention provides a kit of parts
comprising two or more of: 1) an antibody that binds specifically
to LRRK2 phosphorylated at Ser910 or an antibody that binds
specifically to LRRK2 that is not phosphorylated at Ser910; 2) an
antibody that binds specifically to LRRK2 phosphorylated at Ser935,
or an antibody that binds specifically to LRRK2 that is not
phosphorylated at Ser935; 3) a 14-3-3 polypeptide (which may, for
example, be labeled, for example with digoxygenin), or an antibody
that specifically binds to a 14-3-3 polypeptide; and 4) a
fluorescently labeled LRRK2 polypeptide, or polynucleotide encoding
a fluorescently labeled LRRK2.
[0078] A further aspect of the invention provides the use of: 1) an
antibody that binds specifically to LRRK2 phosphorylated at Ser910;
2) an antibody that binds specifically to LRRK2 phosphorylated at
Ser935; 3) a 14-3-3 polypeptide, or an antibody that specifically
binds to a 14-3-3 polypeptide; and/or 4) a fluorescently labeled
LRRK2 polypeptide, or polynucleotide encoding a fluorescently
labeled LRRK2, in a method for assessing the effect of a test
compound on LRRK2 in a cell-based system.
[0079] A further aspect of the invention provides a purified
preparation or kit of parts comprising an LRRK2 polypeptide or
polynucleotide (i.e. a polynucleotide encoding an LRRK2
polypeptide) or antibody binding specifically to LRRK2; and a
14-3-3 polypeptide or polynucleotide (i.e. a polynucleotide
encoding a 14-3-3 polypeptide) or antibody binding specifically to
a 14-3-3 polypeptide. The preparation or kit may, for example,
comprise a recombinant LRRK2 polynucleotide or polypeptide and a
recombinant 14-3-3 polypeptide or polynucleotide. The LRRK2 and
14-3-3 may comprise fluorescent tags suitable for use in a FRET
system, as discussed above. The preparation or kit may comprise
immunoprecipitated LRRK2 polypeptide and 14-3-3 polypeptide. The
preparation or kit may comprise an antibody that specifically binds
to LRRK2 and a 14-3-3 polypeptide (which may, for example, be
labeled, for example with digoxygenin) or an antibody that
specifically binds to a 14-3-3 polypeptide.
[0080] The preparation or kit may be useful in an assay of the
invention.
[0081] By the term "antibody" is included synthetic antibodies and
fragments and variants (for example as discussed above) of whole
antibodies which retain the antigen binding site. The antibody may
be a monoclonal antibody, but may also be a polyclonal antibody
preparation, a part or parts thereof (for example an F.sub.ab
fragment or F(ab').sub.2) or a synthetic antibody or part thereof.
Fab, Fv, ScFv and dAb antibody fragments can all be expressed in
and secreted from E. coli, thus allowing the facile production of
large amounts of the said fragments. By "ScFv molecules" is meant
molecules wherein the V.sub.H and V.sub.L partner domains are
linked via a flexible oligopeptide. IgG class antibodies are
preferred.
[0082] Suitable monoclonal antibodies to selected antigens may be
prepared by known techniques, for example those disclosed in
"Monoclonal Antibodies: A manual of techniques", H. Zola (CRC
Press, 1988) and in "Monoclonal Hybridoma Antibodies: techniques
and Applications", J G R Hurrell (CRC Press, 1982), modified as
indicated above. Bispecific antibodies may be prepared by cell
fusion, by reassociation of monovalent fragments or by chemical
cross-linking of whole antibodies. Methods for preparing bispecific
antibodies are disclosed in Corvalen et al, (1987) Cancer Immunol.
Immunother. 24, 127-132 and 133-137 and 138-143.
[0083] A general review of the techniques involved in the synthesis
of antibody fragments which retain their specific binding sites is
to be found in Winter & Milstein (1991) Nature 349,
293-299.
[0084] By "purified" is meant that the preparation has been at
least partially separated from other components in the presence of
which it has been formed, for example other components of a
recombinant cell. Examples of methods of purification that may be
used are described in the Examples.
[0085] The preparation may be substantially pure. By "substantially
pure" we mean that the said polypeptide(s) are substantially free
of other proteins. Thus, we include any composition that includes
at least 2, 3, 4, 5, 10, 15, 20 or 30% of the protein content by
weight as the said polypeptides, preferably at least 50%, more
preferably at least 70%, still more preferably at least 90% and
most preferably at least 95% of the protein content is the said
polypeptides.
[0086] Thus, the invention also includes compositions comprising
the said polypeptides and a contaminant wherein the contaminant
comprises less than 96, 95, 94, 90, 85, 80 or 70% of the
composition by weight, preferably less than 50% of the composition,
more preferably less than 30% of the composition, still more
preferably less than 10% of the composition and most preferably
less than 5% of the composition by weight.
[0087] The invention also includes the substantially pure said
polypeptides when combined with other components ex vivo, said
other components not being all of the components found in the cell
in which said polypeptides are found.
[0088] A further aspect of the invention includes a method of
characterising an LRRK2 mutant, for example an LRRK2 mutant found
in a patient with Parkinson's Disease, the method comprising the
steps of: a) assessing the phosphorylation state of Ser910 and/or
Ser935 of the LRRK2 mutant; and/or b) assessing the ability of the
LRRK2 mutant to bind a 14-3-3 polypeptide. The method may comprise,
or further comprise, the step of assessing the subcellular location
of the LRRK2 mutant, when expressed in a cell-based system. The
assessing steps may be performed using the antibodies, reagents and
cell-based systems described above.
[0089] All documents referred to herein are hereby incorporated by
reference. For the avoidance of doubt Jaleel et al (2007) Biochem J
405(2), 307-317, PCT/GB2008/001211 and PCT/GB2009/002047 are hereby
incorporated by reference.
[0090] The listing or discussion of an apparently prior-published
document in this specification should not necessarily be taken as
an acknowledgement that the document is part of the state of the
art or is common general knowledge.
[0091] The invention is now described in more detail by reference
to the following, non-limiting, Figures and Examples.
FIGURE LEGENDS
[0092] FIG. 1. Quantitative mass spectrometry identifies 14-3-3 as
a major LRRK2-interactor. 293-HEK cells stably expressing GFP, wild
type full-length GFP-LRRK2 or full-length GFP-LRRK2[G2019S] mutant
were cultured for multiple passages in either R6K4 SILAC media
(GFP-LRRK2 or GFP-LRRK2[G2019S]) or normal ROKO SILAC media (GFP).
Cells were lysed and equal amounts of lysates from GFP and
GFP-LRRK2 (A & C) or GFP and GFP-LRRK2[G2019S] (B & D) were
mixed. Immunoprecipitations were undertaken employing an anti-GFP
antibody and electrophoresed on a SDS-polyacrylamide gel, which was
stained with colloidal blue (A & B). Migration of LRRK2 band is
indicated with an arrowhead and GFP band is indicated with an
arrow. Molecular weights of markers are indicated on the left and
right of the gels. The entire lane from each gel was excised,
digested with trypsin and processed for mass spectrometry. Each
sample was analyzed by Orbitrap mass spectrometry and quantitated
using MaxQuant (version 13.13.10) [28] and a summary of results are
presented in tabular format. The number of peptides and percent of
sequence coverage corresponding to the indicated protein which were
quantitated are shown along with the ratios of enrichment for
labeled versus unlabeled peptides for each comparison of GFP vs.
wild type LRRK2 (C) and GFP vs. LRRK2 [G2019S] (D). The posterior
error probability PEP is shown, which measures the accuracy of
MaxQuant quantitation where the closer to zero the higher the
probability of specific interaction [28].
[0093] FIG. 2. Characterisation of LRRK2 interaction with 14-3-3.
A.) 5 mg of Swiss 3T3 lysate was subjected to immunoprecipitation
with control IgG or anti-LRRK2 (S348C) antibody. Immunoprecipitates
were resolved on 4-12% Novex SDS-polyacrylamide gel and
immunoblotted with antibodies against LRRK2 (S374C), Hsp90 and pan
14-3-3. B.) 5 mg of Swiss 3T3 lysate was subjected to
immunoprecipitation with anti-pan 14-3-3 antibodies and
immunoprecipitates were resolved on 4-12% Novex SDS-polyacrylamide
gels and immunoblotted with antibodies against pan-14-3-3 and LRRK2
(S374C). C.) Lysates of T-Rex HEK 293 FLAG-LRRK2 cells transfected
with pEBG plasmids encoding GST or GST tagged 14-3-3 isoforms and
induced to express LRRK2 by inclusion of 1 .mu.g/ml of doxycycline
in the culture medium. 36 hours post transfection cells were lysed
and glutathione-Sepharose affinity purified proteins immunoblotted
with anti-GST or anti-FLAG antibodies. D.) Fragments encoding the
indicated domains of LRRK2 were transiently expressed in HEK-293
cells and immunoprecipitated with anti-FLAG antibodies. The
immunoprecipitates were resolved on 4-12% Novex SDS-polyacrylamide
gels and probed with either anti-FLAG antibodies or 14-3-3 overlay
with digoxigenin labeled 14-3-3 in a far western assay.
Co-immunoprecipitated 14-3-3 was detected with anti-pan 14-3-3
antibody. E.) Endogenous LRRK2 was immunoprecipitated from Swiss
3T3 cells with anti-LRRK2 (S348C) and subsequently treated with
.lamda.-phosphatase in the absence or presence of EDTA prior to
immunoblot analysis with indicated antibodies or a 14-3-3 overlay
assay. F.) As in E, except experiment undertaken with
immunoprecipitated FLAG-LRRK2 obtained following transient
transfection in HEK-293 cells.
[0094] FIG. 3. Identification of LRRK2 phosphorylation sites, the
sites of 14-3-3 binding and characterization of anti-pS910 and
anti-pS935. A.) Endogenous LRRK2 was immunoprecipitated with
anti-LRRK2 100-500 (S348C) from Swiss 3T3 cells and FLAG-LRRK2 was
immunoprecipitated with anti-FLAG agarose from stable, inducible
T-Rex HEK 293 cells and was resolved on a 4-12% Novex
SDS-polyacrylamide gel and stained with colloidal blue. Gel is
representative of several experiments. LRRK2 tryptic peptides were
subjected to LC-MSMS on an LTQ-Orbitrap mass spectrometer. B.)
Phosphopeptides identified by LTQ-Orbitrap mass spectrometry shown
in tabular format. Observed mass (m/z) and predicted mass (M) are
shown, along with the site of phosphorylation and peptide sequence
identified. The number of experiments evaluated (N) is indicated at
the top of the column and the number of times, in total, the
phosphorylated peptide was identified is indicated. C.) Domain
structure of LRRK2 is presented to scale, with amino acid residues
indicating domain boundaries indicated. Position of identified
phosphorylation sites is shown. D.) The indicated phosphorylation
sites identified in A and B were mutated to Ala and transiently
expressed in HEK-293 cells. LRRK2 was immunoprecipitated with FLAG
agarose and equal amounts of each protein were probed with FLAG
(total) and ability to directly bind 14-3-3 was assessed in an
overlay assay. 14-3-3 and Hsp90 co-immunoprecipitation (Co-IP) was
determined by immunoblotting the immunoprecipitates with pan-14-3-3
and Hsp90 antibodies. Kinase activity was assayed against 30 .mu.M
Nictide and specific activity was determined by correcting
incorporation of phosphate for protein levels in the
immunoprecipitate by quantitative immunoblot using Odyssey LICOR
and is presented as counts per minute/LICOR absorbance units
(cpm/LICOR AU). Data are mean.+-.SEM and were performed in
duplicate and are representative of at least 4 separate
experiments. E.) The indicated forms of LRRK2 were expressed in 293
cells by transient transfection. 36 hours post transfection these
were immunprecipitated with Flag antibody and immunoblotted with
phosphospecific antibodies against S910 (S357C) and S935 (S814C).
Direct binding of immunoprecipitates to 14-3-3 was assessed by
overlay assay and co-immunoprecipitation of 14-3-3 and Hsp90
assessed by immunoblotting with the respective antibodies. F.)
LRRK2 was immunoprecipitated from tissues of wild type male C57BL/6
mice and immunoblotted for Ser910 and Ser 935 phosphyorylation and
14-3-3 binding was assessed by overlay assay as in E. G.) Multiple
sequence alignment of LRRK2 from Homo sapiens (NP.sub.--940980),
Mus musculus (NP.sub.--080006), Rattus norvegicus
(XP.sub.--235581), Bos Taurus (XP.sub.--615760), Canis lupis
familiaris (XP.sub.--543734), and Gallus gallus (XP.sub.--427077).
Position of the phosphorylated residues Serine 910 and 935 are
indicated. Identical residues are indicated. H.) Sequence
comparison of residues surrounding the Ser910 and Ser935
phosphorylation sites of human LRRK2.1.) Multiple sequence
alignment of LRRK2 from Homo sapiens (NP.sub.--940980), Pan
troglodytes (XP.sub.--001168494), Mus musculus (NP.sub.--080006),
Rattus norvegicus (XP.sub.--235581), Bos Taurus (XP.sub.--615760),
Canis lupis familiaris (XP.sub.--543734), and Gallus gallus
(XP.sub.--427077). Position of the phosphorylated residues Serine
910 and 935 are indicated. Identical residues are indicated.
[0095] FIG. 4. H-1152 and sunitinib treatment leads to
dephosphorylation of S910 and 935 and disruption of 14-3-3
interaction. A.) Endogenous LRRK2 was immunoprecipitated with
anti-LRRK2 100-500 (S348C) from Swiss 3T3 cells were treated with
DMSO vehicle control or the indicated concentrations of H-1152 for
90 minutes. Immunoprecipitates were resolved on 4-12% Novex gels
and subjected to 14-3-3 overlay far western analysis and
immunoblotted with anti-pS910 (S357C), anti-pS935 (S814C) and
anti-LRRK2 (S374C) antibodies. Immunoblots were quantitated by
Odyssey LICOR and the amount of LRRK2 phosphorylation is presented
as a ratio of phosphospecific antibody/total LICOR absorbance units
(pS910/LRRK2 [AU]). B.) Endogenous LRRK2 immunoprecipitates were
analyzed as in A, except that cells were treated with H-1152 at 30
.mu.M for the indicated time prior to cell lysis. C.) and D.) as in
A. and B. respectively, except that sunitinib was employed rather
than H1152. Data are mean.+-.SEM and were performed in duplicate
and are representative of at least 2 separate experiments.
[0096] FIG. 5. Evidence that LRRK2 kinase activity controls Ser910
and Ser935 phosphorylation as well as 14-3-3 binding. A & B)
HEK-293 cells transiently expressing the indicated forms of
Flag-LRRK2 were treated with DMSO vehicle control or indicated
concentrations of H1152 or sunitinib for 90 minutes. Cells were
lysed in lysis buffer supplemented with 0.5% NP40 and 150 mM and
subjected to anti-FLAG immunoprecipitation. Immunoprecipitates were
resolved on 4-12% Novex SDS-polyacrylamide gels and subjected to
immunoblot with FLAG (total LRRK2), anti-pS910, anti-pS935 as well
as a 14-3-3 overlay assay. Similar results were obtained in 2
separate experiments.
[0097] FIG. 6. Evidence that Ser910 and Ser935 phosphorylation is
not mediated by LRRK2 autophosphorylation. Endogenous LRRK2 was
immunoprecipitated from Swiss 3T3 cells treated with DMSO or 30
.mu.M H1152 or 10 .mu.M sunitinib for 2 h to induce
dephosphorylation of Ser 910 and Ser 935. Immunoprecipitates were
washed with lysis buffer containing 0.5 M NaCl to remove inhibitor
and were then incubated in kinase buffer containing 20 .mu.M
Nictide with the presence or absence of magnesium-ATP for 30 min.
Following incubation, immunoprecipitates were centrifuged at 8000
rpm for 0.5 min and the supernatant spotted on to P81 paper for
measurement of LRRK2 kinase activity. Sample buffer was added to
the pelleted beads and LRRK2 S910 and S935 phosphorylation was
quantified following immunoblot analysis. A membrane was also
subjected to autoradiography to assess LRRK2 autophosphorylation.
The minor effect that H-1152 had on LRRK2 kinase assay is not
significant.
[0098] FIG. 7. Proposed model of how LRRK2 controls S0er910 and
Ser935 phosphorylation leading to 14-3-3 binding. Our data suggest
that LRRK2 kinase activity stimulates the activity of a protein
kinase or inhibits the activity of a protein phosphatase that acts
on Ser910 and Ser935. This enables LRRK2 to interact with 14-3-3
isoforms and stabilises diffuse cytoplasmic localisation of LRRK2.
Treatment of cells with LRRK2 inhibitors thus leads to
dephosphorylation of Ser910 and Ser935 and dissociation of 14-3-3
isoforms. Our findings indicate that LRRK2 phosphorylation of
Ser910 and Ser935 as well as 14-3-3 binding could be employed as a
biomarker to benchmark efficacy of LRRK2 inhibitors that are being
developed.
[0099] FIG. 8. 14-3-3 binding influences LRRK2 cytoplasmic
localisation A.) Stable-inducible T-REx cells lines harbouring the
indicated forms of LRRK2 were induced for 24 hours with 0.1
.mu.g/ml doxycycline to induce expression of GFP-LRRK2. Equal
amount of cell lysate from induced cells of each mutant was
subjected to immunoblot analysis with anti-GFP antibodies to detect
the fusion protein or anti-GAPDH as a loading control. B.)
Fluorescent micrographs representative of cultures of the indicated
forms GFP-LRRK2 are shown. Cytoplasmic pools of GFP-LRRK2 observed
in the non-14-3-3 binding mutants are indicated with white
arrowheads.
[0100] FIG. 9. Activity and 14-3-3 Binding of 41 Parkinson's
disease associated LRRK2 mutants. The inset illustrates the domain
structure of LRRK2 with the Leucine-Rich Repeats (LRR), Ras of
Complex GTPase domain (ROC), Carboxy terminal of Roc (COR), and
Kinase catalytic domain (Kinase) and the minimal WD40 repeat domain
(WD40) annotated. Positions of the PD associated mutations are
shown. The amino acid boundaries of the domains are indicated. The
indicated variants of full length FLAG tagged LRRK2 were
transiently expressed in HEK 293 cells and subjected to
immunoprecipitation analysis. Kinase activity of immunoprecipitates
was assessed against LRRKtide and specific activity was determined
by quantitative anti-FLAG immunoblot analysis of LRRK2 using LICOR
technology and was defined as cpm/LICOR. Wild type LRRK2 activity
was set to 1 and the mutant activities are relative to wild type.
Assays were performed in duplicate, for three experiments, bars are
s.e.m. FLAG-LRRK2 immunoprecipitates were also subjected to
immunoblot analysis with anti-FLAG, anti-pSer910 and anti-pSer935
antibodies. 14-3-3 binding to the LRRK2 variants was assessed by
14-3-3 far western analyses and 14-3-3 immunoblotting for
co-precipitating 14-3-3.
[0101] FIG. 10. Disruption of Ser910/Ser935 phosphorylation and
14-3-3 binding in LRRK2[R1441C] knockin mice. Brain, kidney and
spleen tissue was rapidly excised from three homozygous
LRRK2[R1441C] knockin mice and three wild type littermate controls
and snap-frozen in liquid nitrogen. LRRK2 was immunoprecipitated
from whole tissue lysate of brain, kidney or spleen.
Immunoprecipitates were immunoblotted for phosphorylation of LRRK2
at Ser910 and Ser935 and for total LRRK2. Ability to interact with
14-3-3 binding was assessed by 14-3-3 far western analysis. Note
insufficient sample from Spleen was available for measuring Ser910
phosphorylation.
[0102] FIG. 11. Localisation of 41 PD associated LRRK2 mutants.
Parallel cultures of stable inducible T-REx cells lines harboring
the indicated mutations were induced for 24 hours with 1 .mu.g/ml
doxycycline to induce expression of GFP-LRRK2. A.) Equal amount of
cell lysate from induced cells of each mutant was subjected to
immunoblot analysis with anti-GFP antibodies to detect the fusion
protein or anti-ERK1 as a loading control. B.) Fluorescent
micrographs representative of cultures of each PD associated mutant
(panels 1-43) are shown. Cytoplasmic pools of GFP-LRRK2 are
indicated with white arrowheads. Localization analyses were
performed in duplicate, on two independently generated stable cell
lines. Larger panels of each of the micrographs shown are presented
in FIG. 12.
[0103] FIG. 12. Summary of the effects of 41 PD associated LRRK2
mutations. Kinase activity relative to wild type, where - indicates
no detectable activity, + equals approximately no change and each
fold increase represented by and additional +. Effects of
phosphoserine 910 and 935 phosphorylation and direct 14-3-3
binding, where no change is represented by ++, + indicates a
decrease in Ser910/Ser935 phosphorylation or 14-3-3 binding and -
indicates no detectable Ser910/Ser935 phosphorylation or 14-3-3
binding. Localization is denoted as diffuse for diffuse cytoplasmic
staining. Aggregate denotes the appearance of cytoplasmic pools. We
have divided LRRK2 mutants into six groups. Group1 mutants display
>2-fold increase in kinase activity, but normal 14-3-3 binding
and diffuse localisation (green shading--*). Group 2 mutants
display normal kinase activity but reduced Ser910/Ser935
phosphorylation as well as 14-3-3 binding and accumulate within
cytoplasmic pools (peach shading--**). Group 3 mutants display
normal kinase activity but reduced Ser910/Ser935 phosphorylation as
well as 14-3-3 binding and exhibit diffuse cytoplasmic localisation
(blue shading--***). The Group 4 mutant displayed normal kinase
activity, Ser910/Ser935 phosphorylation as well as 14-3-3 binding
but accumulate within cytoplasmic pools (yellow shading--****). The
Group 5 mutant displays no kinase activity, Ser910/Ser935
phosphorylation or 14-3-3 binding and localize diffusely (red
shading--*****). Group 6 mutants display properties similar to wild
type LRRK2 (non-shaded).
[0104] FIG. 13. Localization of 41 PD associated LRRK2 mutants.
FIGS. 13.1-13.8 depict the same localisation data as presented in
FIG. 11 (panels 1-43) except that larger panels of each of the
micrographs is presented in FIG. 13 to improve clarity.
[0105] FIG. 14. Disruption of 14-3-3 binding induces accumulation
of LRRK2 within cytoplasmic aggregates. A.) Stable-inducible T-REx
cells lines harboring the indicated forms of LRRK2 were induced for
24 hours with 0.1 .mu.g/ml doxycycline to induce expression of
GFP-LRRK2. The indicated cell lines were treated in the absence or
presence of the indicated dose of H-1152 for 90 min prior to
fixation. Representative fluorescent micrographs of GFP-LRRK2
localisation are shown. Cytoplasmic aggregates of GFP-LRRK2 are
indicated with white arrowheads. B.) Fluorescent micrographs
representative of cultures of the indicated forms GFP-LRRK2 are
shown. Cytoplasmic aggregates of GFP-LRRK2 are indicated with white
arrowheads. Localisation analyses were performed in duplicate, and
similar results observed in two independent experiments.
[0106] FIG. 15. Endogenous LRRK2 was immunoprecipitated with
anti-LRRK2 100-500 (S348C) from Swiss 3T3 cells treated with DMSO
vehicle control or the indicated concentrations of the potent ROCK
inhibitor GSK429286A for 90 minutes. Immunoprecipitates were
subjected to immunoblot analysis with the indicated antibody as
well as 14-3-3 overlay far western analysis. Immunoblot analysis
was quantified by Odyssey LICOR analysis and the amount of LRRK2
phosphorylation is presented as a ratio of phosphospecific
antibody/total LICOR absorbance units (pS910/LRRK2 [AU]).
[0107] FIG. 16. HEK-293 stably expressing GFP-LRRK2 were treated
with DMSO, or the following inhibitors dissolved in DMSO, at the
indicated concentration for 90 minutes. Sunitinib (LRRK2 inhibitor
[40]), GDC-0941 (PI3K inhibitor [41]), PI-103 (Dual mTOR/PI3K
inhibitor [42]), BX-795 (Dual MARK/PDK1 inhibitor [43], AKTi1/2
(PKB inhibitor [44]), KU0063794 (mTOR inhibitor [45]), CHIR-99021
(GSK3 inhibitor [46]), BAY439006 (Raf inhibitor [47]), PD-0325901
(MEK1 inhibitor [48]), BID-1870 (RSK inhibitor [49]), BIRB-0796
(p38 MAPK inhibitor [50]), SB203580 (p38 MAPK inhibitor [51]),
AS601245 (JNK inhibitor [52]), SP600125 (JNK inhibitor [53]),
BMS345541 (IKK inhibitor [54]), PS-1145 (IKK inhibitor [55]), TPL2
inhibitor 31 (Cot/TPL2 inhibitor [56]), Necrostatin (RIPK inhibitor
[57]), H-89 (Dual PKA/ROCK inhibitor [58]), RO-31-8220 (PKC
inhibitor [59]), Rottlerin (PKC inhibitor [60]), STO-609 (CaMKK
inhibitor [61]), Compound C (AMPK inhibitor [62]), AG490 (JAK
inhibitor [63]), PP1 (Src inhibitor [64]), PP2 (Src inhibitor
[65]), GSK429286A (ROCK inhibitor [40]), Harmine (Dual CDK/DYRK
inhibitor [66]), Roscovitine (CDK inhibitor[67]), SU-6668 (Dual
Src/Aurora kinase inhibitor [68]), VX-680 (Aurora kinase inhibitor
[69]), Quercetagetin (PIMK inhibitor [70]), 401 KuDOS (DNAPK
inhibitor [71]), BI-2536 (PLK1 inhibitor [72]). Following lysis 30
.mu.g of lysate was resolved by SDS-PAGE and immunoblotted for
LRRK2 phosphorylation at Ser910 and Ser935. Total LRRK2 was
assessed by GFP immunoblot. The immunoblots shown are
representative of 2 independent experiments.
EXAMPLE 1
Inhibition of Kinase Activity Leads to Dephosphorylation of LRRK2
at Ser910/Ser935 and Disruption of 14-3-3 Binding. Development of a
Cell-Based Assay to Assess LRRK2 Inhibitors
[0108] The Leucine Rich Repeat Protein Kinase-2 (LRRK2) is mutated
in a significant number of Parkinson's disease patients. Since a
common mutation changing Gly2019 to Ser enhances kinase catalytic
activity, small molecule LRRK2 inhibitors might have utility in
treating Parkinson's disease. However, the effectiveness of
inhibitors is difficult to assess, as no physiological substrates
or downstream effectors of LRRK2 have been identified that could be
exploited to develop a robust cell-based assay. Here we demonstrate
that endogenous LRRK2 interacts with endogenous 14-3-3 isoforms.
This interaction is mediated by phosphorylation of conserved Ser910
and Ser935 residues located before the leucine rich repeat domain.
Strikingly, treatment of Swiss 3T3 cells with two structurally
unrelated inhibitors of LRRK2 (H-1152 or sunitinib), induced
dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby
disrupting 14-3-3 interaction. We suggest that H-1152 and sunitinib
induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2
kinase activity; these compounds failed to induce significant
dephosphorylation of a drug resistant LRRK2[A2016T] mutant.
Moreover, consistent with the finding that non-14-3-3 binding
mutants of LRRK2 accumulate within discrete cytoplasmic pools
rather than diffusely localising throughout the cytoplasm, H-1152
causes LRRK2 to accumulate within cytoplasmic pools. These data
indicate that dephosphorylation of Ser910, Ser935 or disruption of
14-3-3 binding and/or monitoring LRRK2 cytoplasmic localisation can
be used as a marker to assess relative efficacy of LRRK2 kinase
inhibitors in vivo. These findings will aid the development of
LRRK2 kinase inhibitors. They will also stimulate further research
to understand how phosphorylation of Ser910 and Ser935 is
controlled by LRRK2 and establish any relationship to development
of Parkinson's disease.
Materials and Methods
Reagents and General Methods.
[0109] Tissue-culture reagents were from Life Technologies.
Glutathione Sepharose 4B was from Amersham Biosciences and
[.gamma.-.sup.32P]-ATP was from Perkin Elmer. P81 phosphocellulose
paper was from Whatman. Pepceuticals synthesized Nictide. The
Flp-in T-REx system was from Invitrogen and stable cell lines,
generated per manufacturer instructions by selection with
hygromycin, have been described previously [8]. Restriction enzyme
digests, DNA ligations and other recombinant DNA procedures were
performed using standard protocols. All mutagenesis was carried out
using the Quick-Change site-directed-mutagenesis kit (Stratagene).
DNA constructs used for transfection were purified from Escherichia
coli DH5a using Qiagen or Invitrogen plasmid Maxi kits according to
the manufacturer's protocol. All DNA constructs were verified by
DNA sequencing, which was performed by The Sequencing Service,
School of Life Sciences, University of Dundee, Scotland, U.K.,
using DYEnamic ET terminator chemistry (Amersham Biosciences) on
Applied Biosystems automated DNA sequencers. H1152 was purchased
from Calbiochem and Sunitinib from LC Laboratories.
Buffers.
[0110] Lysis Buffer contained 50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1
mM EDTA, 1% (w/v) 1 mM sodium orthovanadate, 10 mM
sodium.beta.-glycerophosphate, 50 mM NaF, 5 mM sodium
pyrophosphate, 0.27 M sucrose, 1 mM Benzamidine and 2 mM
phenylmethanesulphonylfluoride (PMSF) and was supplemented with
either 1% (v/v) Triton X-100 or 0.5% (v/v) NP-40 with 150 mM NaCl
as indicated. Buffer A contained 50 mM Tris/HCl, pH 7.5, 50 mM
NaCl, 0.1 mM EGTA and 0.1% (v/v) 2-mercaptoethanol, and 0.27 M
sucrose. Lambda phosphatase reactions were carried out in buffer A
supplemented with 1 mM MNCl.sub.2 and 2 mM DTT.
Cell Culture, Treatments and Cell Lysis.
[0111] HEK-293 and Swiss 3T3 cells were cultured in Dulbecco's
Modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM
glutamine and 1.times.antimycotic/antibiotic solution. T-REx cell
lines were cultured in DMEM supplemented with 10% FBS and 2 mM
glutamine, 1.times.antimycotic/antibiotic [pen/strep], and 15
.mu.g/ml blastocidin and 100 .mu.g/ml hygromycin. Cultures were
induced to express the indicated protein by inclusion of 1 .mu.g/ml
doxycycline in the culture medium for the indicated times or 24
hours.
[0112] Cell transfections were performed by the polyethylenimine
method [12]. Where inhibitors are utilized, they were dissolved in
DMSO and used at the indicated concentrations with an equivalent
volume of DMSO used as a control. The final concentration of DMSO
in the culture medium was never more than 0.1% (v/v). Inhibitors
were added to the culture medium for the indicated times before
lysis. Per 15 cm dish, HEK 293 cells were lysed with 1.0 ml and 3T3
cells were lysed with 0.6 ml of lysis buffer supplemented with the
indicated detergent and clarified by centrifugation at
16,000.times.g at 4.degree. C. for 10 minutes. After induction and
inhibitor treatment, T-REx-GFP expressing cells were lysed at room
temperature with SDS lysis buffer after washing with PBS. SDS
lysates were boiled and sonicated to reduce viscosity. When not
used immediately, all lysate supernatants were snap frozen in
liquid nitrogen and stored at -80.degree. C. until use. Protein
concentrations were determined using the Bradford method with BSA
as the standard.
Antibodies.
[0113] Anti-LRRK2 100-500 (S348C and 5406C) and Anti-LRRK2
2498-2514 (S374C) were described previously [8]. Antibody against
LRRK2 phosphoserine 910 (S357C) was generated by injection of the
KLH conjugated phosphopeptide VKKKSNpSISVGEFY (where pS is
phosphoserine) into sheep and was affinity purified by positive and
negative selection against the phospho and de-phospho peptides
respectively. Antibody against LRRK2 phosphoserine 935 (S814C) was
generated by injection of the KLH conjugated phosphopeptide
NLQRHSNpSLGPIFDH (where pS is phosphoserine) into sheep and was
affinity purified by positive and negative selection against the
phospho and de-phospho peptides respectively. Sheep polyclonal
antibody S662B was raised against MBP-MYPT chicken amino acids
(714-1004). Rabbit polyclonal antibody against MYPT
phosphothreonine 850 was from Upstate (#36-003). Anti GFP antibody
(S268B) was raised against recombinant GFP protein and affinity
purified against the antigen. Anti-FLAG M2 antibody and affinity
matrix were from Sigma (A2220). Nanotrap GFP binder affinity matrix
was from ChromoTek. Rabbit polyclonal antibody recognizing 14-3-3
(K-19, SC-629) and control rabbit IgG (SC-2027) antibody were from
SantaCruz biotechnology. HSP90 antibody was from Cell signalling
technology (#4877). Anti-MARK3 was from Upstate (#05-680).
Immunological Procedures.
[0114] Cell lysates (10-30 .mu.g) were resolved by electrophoresis
on SDS polyacrylamide gels or Novex 4-12% gradient gels, and
electroblotted to nitrocellulose membranes. Membranes were blocked
with 5% skimmed milk (w/v) in 50 mM Tris/HCl, pH 7.5, 0.15 M NaCl
and 0.1% (v/v) Tween (TBST Buffer). For phospho-antibodies, primary
antibody was used at a concentration of 1 .mu.g/ml, diluted in 5%
skimmed milk in TBST with the inclusion of 10 .mu.g/ml
dephosphorylated-peptide. All other antibodies were used at 1
.mu.g/ml in 5% (w/v) milk in TBST. Detection of immune-complexes
was performed using either fluorophore conjugated secondary
antibodies (Molecular Probes) followed by visualisation using an
Odyssey LICOR or by horseradish-peroxidase-conjugated secondary
antibodies (Pierce) and an enhanced-chemiluminescence reagent. For
immunoprecipitations, antibody was non-covalently coupled to
protein G-Sepharose at a ratio of 1 .mu.g antibody/.mu.l of beads,
or anti-FLAG M2-agarose was utilized. Cell lysate was incubated
with coupled antibody for 1 hour. Immune complexes were washed
twice with lysis buffer supplemented with 0.3 M NaCl and twice with
Buffer A. Precipitates were either used as a source of kinase or
immediately analyzed by immunoblot. Digoxigenen (DIG) labelled
14-3-3 for use in overlay far western analysis was prepared as
described in [13]. To directly assess 14-3-3 interaction with
LRRK2, immunoprecipitates were electroblotted to nitrocellulose
membranes and blocked with 5% skimmed milk for 30 minutes. After
washing with TBST, membranes were incubated with DIG labelled
14-3-3 diluted to 1 .mu.g/ml in 5% BSA in TBST overnight at
4.degree. C. DIG 14-3-3 was detected with HRP labelled anti-DIG Fab
fragments (Roche).
SILAC Media.
[0115] SILAC DMEM (high glucose without NaHCO.sub.3, L-glutamine,
arginine, lysine and methionine Biosera #A0347) was prepared with
10% dialyzed FBS (Hyclone) and supplemented with methionine,
glutamine, NaHCO.sub.3, labeled or unlabeled arginine and lysine.
Cells harboring GFP tagged proteins were cultured in SILAC DMEM for
three passages at a 1:10 ratio with the following isotopic
labeling. For GFP versus wild type LRRK2, L-arginine (84 .mu.g/ml;
Sigma-Aldrich) and L-lysine (146 .mu.g/ml lysine; Sigma-Aldrich)
were added to the GFP "light" media, while L-arginine .sup.13C and
L-lysine .sup.13C (Cambridge Isotope Laboratory) were added to the
GFP-LRRK2 wild type "heavy" media at the same concentrations. For
GFP versus LRRK2 G2019S experiments, L-arginine and L-lysine were
added to the GFP "light" media and L-arginine .sup.13C/.sup.15N and
L-lysine .sup.13C/.sup.15N (Cambridge Isotope Laboratory) to the
GFP-LRRK2 G2019S "heavy" media. The amino acid concentrations are
based on the formula for normal DMEM (Invitrogen). Once prepared,
the SILAC media was mixed well, filtered through a 0.22-.mu.m
filter (Millipore). Metabolically labeled cells were induced to
express GFP or the GFP-LRRK2 fusion protein for 24 hours by
inclusion of doxycycline in the culture media.
SILAC Mass Spectrometry.
[0116] Cells metabolically labeled and induced to express either
GFP or LRRK2-wild type or G2019S were lysed in lysis buffer
supplemented with 1% Triton X-100 at 0.5 ml per 10 cm dish. For
each condition individually, 9 mg of cell lysate was subjected to
individual immunoprecipitation with a 20 .mu.l bed volume of GFP
binder agarose beads for 1 hour at 4.degree. C. Beads were washed
once with 5 ml and then with 10 ml of lysis buffer supplemented
with 1% Triton-X 100 and 300 mM NaCl. Beads were then washed once
with 5 ml and then once with 10 ml storage buffer. Bead associated
proteins were eluted with 1.times. LDS sample buffer for 10 min at
70.degree. C. then passed through a 0.22 .mu.m spin-X column.
Control GFP eluates were combined with either eluates of wild type
LRRK2 or LRRK2 G2019S in equal amounts and reduced and alkylated as
above. Samples were resolved on a 12% Novex gel for only one half
of the gel. Gels were stained with colloidal blue overnight and
destained for 3 hours. The entire lane was excised in 9 total bands
and digested with trypsin as described previously [30].
Mass Spectrometry Analysis of Peptides.
[0117] The digests were separated on a Biosphere C.sub.18 trap
column (0.1 mm id.times.2 mm, Nanoseparations, Holland) connected
to a PepMap C18 nano column (75 .mu.m.times.15 cm, Dionex
Corporation) fitted to a Proxeon Easy-LC nanoflow LC-system
(Proxeon, Denmark) with solvent A (2% acetonitrile/0.1% formic
acid/98% water) and solvent B (90% acetonitrile/10% water/0.09%
formic acid). 10 .mu.l of sample (a total of 2 pg of protein) was
loaded with a constant flow of 7 .mu.l/min onto the trap column in
solvent A and washed for 3 min at the same flow rate. After trap
enrichment, peptides were eluted with a linear gradient of 5-50%
solvent B over 90 min with a constant flow of 300 nl/min. The HPLC
system was coupled to a linear ion trap-orbitrap hybrid mass
spectrometer (LTQ-Orbitrap XL, Thermo Fisher Scientific Inc) via a
nanoelectrospray ion source (Proxeon Biosystems) fitted with a 5 cm
Picotip FS360-20-10 emitter. The spray voltage was set to 1.2 kV
and the temperature of the heated capillary was set to 200.degree.
C. Full scan MS survey spectra (m/z 350-1800) in profile mode were
acquired in the Orbitrap with a resolution of 60,000 after
accumulation of 500,000 ions. The five most intense peptide ions
from the preview scan in the Orbitrap were fragmented by
collision-induced dissociation (normalized collision energy 35%,
activation Q 0.250 and activation time 30 ms) in the LTQ after the
accumulation of 10,000 ions. Maximal filling times were 1,000 ms
for the full scans and 150 ms for the MS/MS scans. Precursor ion
charge state screening was enabled and all unassigned charge states
as well as singly charged species were rejected. The lock mass
option was enabled for survey scans to improve mass accuracy. Data
were acquired using the Xcalibur software.
Mass Spectrometry Data MaxQuant Analysis.
[0118] The raw mass spectrometric data files obtained for each
experiment was collated into a single quantitated dataset using
MaxQuant (version 1.0.13.13) (http://www.maxquant.org) and the
Mascot search engine (Matrix Science, version 2.2.2) software.
Enzyme specificity was set to that of trypsin, allowing for
cleavage N-terminal to proline residues and between aspartic acid
and proline residues. Other parameters used within the software:
Variable modifications--Methionine Oxidation;
Database--target-decoy human MaxQuant (ipi.HUMAN.v3.52.decoy)
(containing 148,380 database entries); Labels--R6K4 [for GFP versus
wild type LRRK2] or R10K8 [for GFP versus LRRK2 G2019S]; MS/MS
tolerance-0.5 Da; (e) Top MS/MS peaks per 100 Da-5; Maximum missed
cleavages-2; Maximum of labeled amino-acids: 3; False Discovery
Rate (FDR): 1%.
LRRK2 Immunoprecipitation Kinase Assays.
[0119] Peptide Kinase Assays were set up in a total volume of 50
.mu.l with immunoprecipitated LRRK2 as a source of kinase, in 50 mM
Tris pH 7.5, 0.1 mM EGTA, 10 mM MgCl.sub.2 and 0.1 mM
[.gamma.-.sup.32P]ATP (.about.500-1000 cpm/pmol) in the presence of
30 .mu.M Nictide peptide substrate. Reactions were terminated by
applying 30 .mu.l of the reaction mixture on to P81
phosphocellulose paper and immersion in 50 mM phosphoric acid.
After extensive washing, reaction products were quantitated by
Cerenkov counting. One half of the remaining reaction was subjected
to immunoblot analysis using the Odyssey LICOR system and specific
activity is represented as cpm/LICOR independent density
values.
[0120] 500 .mu.g of transfected cell lysates was subjected to
immunoprecipitation with 5 .mu.l bed volume of anti-FLAG agarose
for 1 hr. Beads were washed twice with Lysis Buffer supplemented
with 300 mM NaCl, the twice with Buffer A. Peptide Kinase Assays
were set up in a total volume of 50 .mu.l with immunoprecipitated
LRRK2 in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10 mM MgCl2 and 0.1 mM
[.gamma.-.sup.32]ATP (-300-500 cpm/pmol) in the presence of 200
.mu.M long variant of the LRRKtide peptide substrate
(RLGRDKYKTLRQIRQGNTKQR) [9, 10] or the Nictide peptide substrate
(RLGWWRFYTLRRARQGNTKQR) [10]. Reactions were terminated by applying
30 .mu.l of the reaction mixture on to P81 phosphocellulose paper
and immersion in 50 mM phosphoric acid. After extensive washing,
reaction products were quantitated by Cerenkov counting. One half
of the remaining reaction was subjected to immunoblot analysis
using the Odyssey LICOR system and specific activity is represented
as cpm/LICOR independent density values
Phosphorylation Site Identification by Mass Spectrometry.
[0121] Endogenous and recombinant LRRK2 was immunoprecipitated from
50 mg of Swiss 3T3 lysate or T-Rex cells induced to express
FLAG-LRRK2 cell lysate using anti-LRRK2 (100-500) or anti-FLAG
agarose, respectively. Immunoprecipitates were eluted from the
affinity matrices using 2.times.LDS sample buffer or 200 .mu.g/ml
FLAG peptide then filtered through a 0.2 .mu.m Spin-X column
(Corning) before reduction with 10 mM dithiothretol and alkylation
with 50 mM iodoacetamide. Samples were heated for 10 min at
70.degree. C. and resolved on 4-12% Novex gels before staining with
colloidal blue (Invitrogen). Bands corresponding to LRRK2 were
excised and digested with trypsin as described previously [30].
Samples were analyzed on an LTQ Orbitrap XL mass spectrometer
(Thermo) as described above, except the top 5 ions were fragmented
in the linear ion trap using multistage activation of the neutral
loss of phosphoric acid from the parent ion (neutral loss
masses=49, 32.33 and 24.5 for z=2, 3 and 4). Mascot generic files
were created from the raw files using raw2 msm (gift from M.Mann)
and were searched on a local Mascot server (matrixscience.com)
using the International Protein Index (IPI) mouse database for
endogenous LRRK2 or the IPI human database for recombinant
LRRK2.
Fluorescence Microscopy.
[0122] HEK-293 Flp-in T-REx were purchased from Invitrogen and
stable cells harbouring GFP tagged wild type and mutant forms of
LRRK2 were generated using standard protocols. Cells were plated in
4-well glass bottom, CC2 coated chamber slides (Nunc). One day
after plating, cells were induced with 0.1 .mu.g/ml doxycycline and
24 hr later, cells were fixed in 4% paraformaldehyde buffered in
phosphate buffered saline (purchased from USB, #19943). Cells were
mounted in ProLong Gold (Invitrogen) and imaged under the same
settings for each mutant, on a Zeiss LSM 700 confocal microscope
using an a Plan-Apochromat x100 objective.
Results
[0123] Association of LRRK2 with 14-3-3.
[0124] We employed quantitative Stable Isotope Labelling with Amino
acids in Cell culture (SILAC)-based mass spectrometry to identify
proteins associated with immunoprecipitates of stably expressed
full length GFP-LRRK2 (FIGS. 1A & 1B) as well as the
GFP-LRRK2[G2019S] mutant (FIGS. 1C & 1D) derived from HEK-293
cells. The top hit, that was enriched at 10 to 30-fold higher
levels with GFP-LRRK2 or GFP-LRRK2[G2019S] compared to GFP alone,
comprised beta, eta, theta, zeta and epsilon isoforms of 14-3-3 for
wild type LRRK2 (FIG. 1B) and beta, theta, zeta, gamma and epsilon
isoforms for LRRK2 [G20195] (FIG. 1D). The two other major
interactors that were observed comprised two isoforms of the heat
shock protein-90 (Hsp90) chaperone-associated with their
kinase-specific targeting CDC37 subunit (enriched 5 to 15-fold).
Hsp90 and CDC37 associated with both wild type LRRK2 as well as
LRRK2[G2019S] mutant and have previously been reported to interact
with LRRK2 [14]. No other significant interactors of LRRK2 were
observed in our interactor screens.
[0125] We found that endogenous 14-3-3 as well as Hsp90 was
co-immunoprecipitated with endogenous LRRK2 from Swiss 3T3 cells
(FIG. 2A). We also observed that endogenous LRRK2 was
co-immunoprecipitated with an antibody that recognises endogenous
14-3-3 isoforms from Swiss 3T3 cells (FIG. 2B). Plasmids encoding
for the expression of all seven isoforms of human 14-3-3 were
transfected into previously generated HEK-293 cells stably
expressing full length FLAG-LRRK2 [8]. Following affinity
purification, apart from atypical sigma isoform, all other forms of
14-3-3 interacted with FLAG-LRRK2 (FIG. 1C). We also observed that
whilst full length LRRK2 associated with endogenous 14-3-3 in 293
cells, in parallel experiments, various isolated domains of LRRK2
tested, failed to bind 14-3-3 (FIG. 1D). This observation was
confirmed employing a 14-3-3 overlay-far western binding assay,
where full length LRRK2, but not isolated domains bound to
digoxoxigenin-labelled 14-3-3 (FIG. 1D-lower panel).
[0126] 14-3-3 isoforms mostly interact with specific phosphorylated
residues on their binding partners [11, 15]. To verify whether
association of 14-3-3 with LRRK2 was dependent upon
phosphorylation, we incubated endogenous LRRK2 (FIG. 2E) or
overexpressed FLAG-LRRK2 (FIG. 2F) in the presence or absence of
lambda phosphatase. Treatment of endogenous or overexpressed LRRK2
with lambda phosphatase markedly reduced interaction of 14-3-3
assessed using the overlay assay. Inclusion of EDTA in the assay,
which inhibits the lambda phosphatase, prevented lambda phosphatase
from suppressing 14-3-3 binding to LRRK2 (FIGS. 2E & 2F).
Residual binding of 14-3-3 to LRRk2 following lambda phosphatase
treatment is presumably due to incomplete dephosphorylation of
LRRK2.
Mapping of Major Phosphorylation Sites on Endogenous LRRK2.
[0127] To determine which phosphorylated residue(s) mediate binding
to 14-3-3, we performed detailed phospho-peptide orbitrap mass
spectrometry analysis of endogenous LRRK2 immunoprecipitated from
mouse Swiss 3T3 cells (FIG. 3A). This revealed three clear
phosphorylation sites namely Ser860, Ser910 and Ser935 (FIG. 3B).
These residues lie in the N-terminal non-catalytic region of LRRK2
just prior to the leucine rich repeats (FIG. 3C). We also analysed
phosphorylation of overexpressed full length human FLAG-LRRK2
expressed in HEK-293 cells, which confirmed that Ser860, Ser910 and
Ser935 were major sites of phosphorylation (FIGS. 3A & 3B). In
addition, we found three other phosphorylation sites in the
overexpressed human FLAG-LRRK2 preparation namely Ser955, Ser973
and Ser976 (FIGS. 3B & 3C). The phospho-peptides encompassing
Ser955, Ser973 and Ser976 were also detected in our analysis of
endogenous LRRK2 but due to the lower abundance of these peptides
we were unable to assign the exact phosphorylation sites (data not
shown).
Phosphorylation of Ser910 and Ser935 Mediates 14-3-3 Binding, but
does not Control Kinase Activity.
[0128] We observed that mutation to Ala of Ser860, Ser955, Ser973,
Ser976 or both Ser973+976 phosphorylation sites, did not affect
binding of 14-3-3 to full length FLAG-LRRK2 (FIG. 3D). Strikingly
however, mutation of Ser910 and/or Ser935 to Ala, ablated
interaction, indicating that phosphorylation of these residues
mediates binding of LRRK2 to 14-3-3 isoforms (FIG. 3D). Mutations
of the identified phosphorylation sites did not affect protein
kinase activity of LRRK2 as measured against the Nictide substrate
peptide (FIG. 3D).
[0129] We next generated phosphospecific antibodies recognising
LRRK2 phosphorylated at Ser910 or Ser935. These antibodies were
specific, as mutation of Ser910 to Ala ablated recognition of LRRK2
with phospho-Ser910 antibody and similarly, mutation of Ser935
abolished recognition with the phospho-Ser935 antibody (FIG. 3E).
We consistently observed that mutation of Ser910 to Ala reduced
phosphorylation of Ser935 about two-fold and vice versa mutation of
Ser935 reduced phosphorylation of Ser910 around two-fold as
quantitated by LICOR (FIG. 3E). Utilising these antibodies, we
demonstrate that endogenous LRRK2 immunoprecipitated from mouse
brain, kidney and spleen was phosphorylated at Ser910 as well as
Ser935 and also bound 14-3-3 (FIG. 3F).
[0130] Sequence alignments indicate that the Ser910 and Ser935
sites as well as residues surrounding them are highly conserved in
mammalian species (FIG. 3G). This region encompassing Ser910 and
Ser935 is not present in Caenorhabditis elegans or Drosophila
melanogaster LRK-1, or indeed mammalian LRRK1. Comparison of the
residues surrounding Ser910 and Ser935 indicates some striking
similarities (FIG. 3 H i.e. basic residues -3 and -4 positions, Ser
residue at the -2 position, Asn at the -1 position and a large
hydrophobic residue at the +1 position).
LRRK2 Inhibitors Induced Dephosphorylation of Ser910/935 and
Disrupted 14-3-3 Binding.
[0131] Incubation of Swiss 3T3 cells with increasing amounts of the
LRRK2 inhibitors H-1152 (FIG. 4A) or sunitinib (FIG. 4C) resulted
in a dose dependent dephosphorylation of endogenous LRRK2 at Ser910
and Ser935 which was accompanied by a concomitant reduction in
14-3-3 binding. 10-30 .mu.M H-1152 or 3-10 .mu.M sunitinib induced
almost complete dephosphorylation of Ser910 and Ser935 resulting in
a loss of 14-3-3 binding. The inhibitory effects of H-1152 (FIG.
4B) and sunitinib (FIG. 4D) on endogenous LRRK2-Ser910/Ser935
phosphorylation and 14-3-3 binding were observed within 30 min and
sustained for at least 2 hours.
Evidence that LRRK2 Kinase Activity Controls Ser910 and Ser935
Phosphorylation as Well as 14-3-3 Binding.
[0132] To determine whether the effect of H1152 and sunitinib on
LRRK2 phosphorylation and 14-3-3 binding resulted from inhibition
of LRRK2 protein kinase activity, we treated HEK-293
over-expressing LRRK2[G2019S] or the H1152/Sunitinib resistant
LRRK2[A2016T+G2019S] mutant with LRRK2 inhibitors. As observed with
the endogenous LRRK2, we found that H-1152 and sunitinib induced a
dose-dependent dephosphorylation of the Parkinson's disease
LRRK2[G2019S] mutant at Ser910 and Ser935 as well as disrupting
binding to 14-3-3 (FIG. 5A-upper panel). Crucially however, neither
H-1152 nor sunitinib significantly inhibited Ser910 or Ser935
phosphorylation or 14-3-3 binding to drug resistant
LRRK2[A2016T+G2019S] mutant (FIG. 5A-lower panel). This strongly
suggests that the ability of H1152 and sunitinib to induce
dephosphorylation of Ser910 as well as Ser935 and hence disrupt
14-3-3 binding is dependent upon the ability of these compounds to
inhibit LRRK2 protein kinase activity.
[0133] In agreement with the pharmacological data demonstrating
that H-1152 and sunitinib inhibit mutant LRRK2[G2019S] 2 to 4-fold
more potently than wild type LRRK2 [8], we found that H1152 and
sunitinib were more potent in inducing dephosphorylation and
impairing binding to 14-3-3 to LRRK2[G2019S] than wild type LRRK2
(compare FIG. 5A & FIG. 5B-upper panels). The potency of H-1152
and sunitinib at inducing dephosphorylation of wild type FLAG-LRRK2
in 293 cells was similar to the effects of these drugs observed for
endogenous LRRK2 in Swiss 3T3 cells (compare FIGS. 4 and 5B).
Evidence that LRRK2 does not Autophosphorylate Ser910 and
Ser935.
[0134] LRRK2 possesses marked preference for phosphorylating
threonine over serine residues [8], suggesting that Ser910 and
Ser935 phosphorylation might not be mediated by
autophosphorylation. Consistent with this, other studies
investigating LRRK2 autophosphorylation sites have mapped a number
of phospho-threonine autophosphorylation sites, but not reported
LRRK2 to phosphorylate at Ser910 or Ser935 [16-18]. To further
investigate whether endogenous LRRK2 can phosphorylate itself at
Ser910 and Ser935, we treated Swiss 3T3 cells with either no drug,
or 30 .mu.M H-1152 in order to induce dephosphorylation of Ser910
and Ser935 (FIG. 6). Endogenous LRRK2 was immunoprecipitated,
washed to remove drug and immunoprecipitates were incubated in the
absence or presence of magnesium-ATP. After 30 min, LRRK2 kinase
activity as well as phosphorylation of Ser910 and Ser935 was
quantified. These studies revealed that the LRRK2 isolated from
H-1152 treated cells was dephosphorylated, and possessed the same
activity as LRRK2 isolated from untreated cells indicating that the
drug had been removed (FIG. 6). Importantly, we observed no
increase in phosphorylation of Ser910 or Ser935 following
incubating LRRK2 from H1152 treated cells with magnesium-ATP. The
amount of phosphorylation of LRRK2 isolated from non-drug treated
cells on Ser910 and Ser935 was also not increased in the
autophosphorylation reaction.
Effect of Multiple Signal Transduction Inhibitors on Ser910/Ser935
Phosphorylation.
[0135] To gain further insight into the specificity of Ser910 and
Ser 935 dephosphorylation HEK293 cells stably expressing GFP-LRRK2
were treated with a panel of 33 kinase inhibitors including those
that suppress major signal transduction pathways in cells including
PI 3-kinase, mTOR, ERK, p38, JNK and innate immune signalling
pathways (FIG. 16). All inhibitors were utilised at the higher
limits of concentrations routinely employed in the literature and
our unit known to suppress signalling pathways. As expected, 10
.mu.M Sunitinib induced marked dephosphorylation of Ser910 and Ser
935 whilst 32 of the inhibitors tested did not significantly affect
dephosphorylation of Ser910/Ser935. Some dephosphorylation of
Ser910 and Ser935 was observed with the relatively non specific JNK
inhibitor SP600125 which was used at a concentration of 15 .mu.M
and is known to inhibit many protein kinases more potently than JNK
[34]. It should be noted that the more potent JNK inhibitor
AS601245 did not induce dephosphorylation of these sites. Further
work will be required to delineate the protein kinase(s) that
directly mediate Ser910 and Ser935 phosphorylation.
Disruption of 14-3-3 Binding Alters Cellular Localisation of
LRRK2.
[0136] A common role of 14-3-3 proteins is to influence the
subcellular localisation of the protein to which it binds. We
therefore studied whether 14-3-3 binding might affect LRRK2
cellular localisation. To ensure low level and as uniform as
possible expression, we generated Flp-in T-REx 293 cells that
stably express wild type and non-14-3-3-binding Ser910/Ser935
mutant forms of full-length GFP-LRRK2. Immunoblot analysis revealed
that wild type and mutant GFP-LRRK2 forms were expressed at similar
levels (FIG. 8A). We next studied the cellular localisation using
confocal microscopy and found, that consistent with a previous
report [18], wild type LRRK2 was uniformly distributed throughout
the cytosol and excluded from the nucleus (FIG. 8B). In contrast,
the non-14-3-3-binding LRRK2[S910A], LRRK2[S935A] and
LRRK2[S910A+S935A] mutants accumulated within cytosolic pools (FIG.
8B).
Characterisation of 14-3-3 Binding of 41 LRRK2 Disease Associated
Mutants.
[0137] We next decided to investigate the Ser910/Ser935
phosphorylation and 14-3-3 binding properties of 41 Parkinson's
disease forms of LRRK2. The location of the different mutations in
LRRK2 analysed is indicated in the FIG. 9 inset. We expressed
full-length wild type and mutant forms of LRRK2 with an N-terminal
Flag epitope tag in 293 cells. LRRK2 was immunoprecipitated and
levels of protein determined by quantitative LICOR-immunoblotting
analysis. Similar levels of LRRK2 forms were subjected to
immunoblot analysis, which revealed that most of the mutants were
phosphorylated at Ser910 and Ser935 to a similar extent as the wild
type enzyme and interacted with 14-3-3 (FIG. 9, lower panel).
However, Ser910/Ser935 phosphorylation and hence 14-3-3 binding
were abolished in four mutants (R1441G, Y1699C, E1874stop and
12020T) (FIG. 9, lower panel). Phosphorylation of Ser910/Ser935 and
14-3-3 binding were significantly reduced in six other mutants
(M712V, R1441H, R1441c, A1442P, L1795F and G2385R) (FIG. 9, lower
panel).
[0138] We also compared the relative protein kinase specific
activity of the 41 mutant forms of LRRK2 employing the LRRKtide
peptide substrate [9] (FIG. 5). Consistent with previous work
[7-9], LRRK2[G2019S] mutant possessed .about.3-fold higher specific
activity than wild type LRRK2. Two other mutants LRRK2[R1728H] and
LRRK2[T2031S] also exhibited two and four-fold increased activity
respectively than wild type LRRK2. Apart from the R1874stop
mutation that lacks the kinase domain and is therefore inactive,
all other mutants tested possessed similar activity to wild type
LRRK2.
Association of 14-3-3 with Endogenous LRRK2 is Impaired in
LRRK2[R1441C] Knockin Mice.
[0139] To obtain further evidence that the LRRK2[R144C] Parkinson's
disease mutation disrupts 14-3-3 binding, we compared levels of
14-3-3 associated with endogenous LRRK2 derived from previously
reported littermate wild type and homozygous LRRK2[R144C] knockin
mice [19]. LRRK2 was immunoprecipitated from spleen, kidney and
brain from three separate mice of each genotype. Immunoblotting and
14-3-3 overlay analysis demonstrated that level of LRRK2 expression
was similar in the wild type and knock-in mice, however the level
of Ser910/Ser935 phosphorylation and associated 14-3-3 was markedly
reduced in tissues derived from LRRK2[R1441C] knock-in mice
compared to wild type (FIG. 10). The largest effect of the mutation
was observed in kidney.
Cellular Localisation of 41 Mutant Forms of LRRK2.
[0140] To ensure low level and as uniform as possible expression of
wild type and mutant forms of full length GFP-LRRK2, we generated
the Flp-in T-REx 293 cells that stably express wild type and mutant
forms of GFP-LRRK2. Immunoblot analysis revealed that GFP-LRRK2
forms were expressed at relatively similar levels (FIG. 11A). The
localisation of wild type, kinase dead and many other LRRK2 mutants
studied were uniformly distributed throughout the cytosol and
excluded from the nucleus with no accumulation within cytoplasmic
pools observed (see FIG. 11B or FIG. 13 to view larger images).
Strikingly, most mutants that displayed reduced Ser910/Ser935
phosphorylation and binding to 14-3-3, accumulated within cytosolic
pools (R1441c, R1441G, R1441H, A1442P, Y1699C, L1795F, 12020T). Not
counting the truncated E1874stop mutant, which displays diffuse
cytoplasmic localisation, only two other mutants (M712V and G2385R)
displaying reduced Ser910/Ser935 phosphorylation and 14-3-3 binding
(FIG. 9) but did not accumulate in cytoplasmic pools (FIG. 11B or
FIG. 13). Only a single mutant (R1067Q) was found to interact with
14-3-3 and accumulate within cytoplasmic pools (FIG. 11B or FIG.
13).
Disruption of 14-3-3 Binding Alters Cellular Localisation of
LRRK2.
[0141] Mutants of LRRK2 that do not interact with 14-3-3 rather
than being diffusely localised throughout the cytoplasm accumulate
within cytoplasmic aggregates, as shown in this Example. This
prompted us to investigate whether H-1152 treatment induces
cytoplasmic re-localisation of GFP-LRRK2 or GFP-LRRK2[G2019S] to
discrete cytoplasmic pools (FIG. 14). We employed stable-inducible
T-REx cells lines expressing at low levels drug sensitive or drug
resistant (A2016T mutant) forms of GFP-LRRK2 or GFP-LRRK2[G2019S].
In untreated cells, GFP-LRRK2 and GFP-LRRK2[G2019S] was diffusely
localised throughout the cytoplasm and not observed in the nucleus
(FIG. 14A). However, H-1152 treatment induced a marked accumulation
of LRRK2 within cytoplasmic pools (FIG. 14A). In accordance with
this effect of H-1152 being mediated via inhibition of LRRK2 kinase
activity, no cytoplasmic pool accumulation was observed upon
treatment of cells expressing drug resistant LRRK2[A2016T] or
LRRK2[A2016T+G2019S] with H-1152 (FIG. 4A). As a further control,
we studied the localisation of GFP-LRRK2[R1441C] and
GFP-LRRK2[Y1699C] that do not bind 14-3-3 and were previously shown
to accumulate within cytoplasmic pools [this Example; 33]. We
confirmed that these mutants accumulated within cytoplasmic
pools-like structures similar to those observed for GFP-LRRK2 and
GFP-LRRK2[G2019S] following treatment with H-1152 (compare FIG. 14A
& FIG. 14B).
Discussion
[0142] The key finding in this paper is that the kinase activity of
LRRK2 indirectly controls phosphorylation of Ser910/Ser935 and
hence 14-3-3 binding as well as LRRK2 cytoplasmic localisation. In
the cell lines we have investigated (Swiss 3T3 (FIG. 4) and HEK-293
(FIG. 5)), phosphorylation of LRRK2 at Ser910 and Ser935 and hence
binding to 14-3-3 was reversed by treatment with the structurally
diverse H-1152 and sunitinib LRRK2 inhibitors. We have also found
that H-1152 induces LRRK2 to accumulate within discrete cytoplasmic
pools that are similar to those observed for LRRK2 mutants that do
not bind 14-3-3 (FIG. 14B). We conclude that dephosphorylation and
cytoplasmic re-localisation results from inhibition of LRRK2 kinase
activity, as LRRK2 inhibitors are ineffective at inducing
dephosphorylation or re-localisation of drug resistant
LRRK2[A2016T] mutants (FIG. 5). The finding that, H-1152 and
sunitinib are more potent at inducing Ser910/Ser935
dephosphorylation of LRRK2[G2019S] than wild type LRRK2 (FIG. 5),
is also consistent with these drugs inhibiting LRRK2[G2019S] two to
four-fold more potently than the wild type LRRK2 [8].
[0143] We demonstrate that 14-3-3 isoforms interact with endogenous
LRRK2 and this is mediated by phosphorylation of Ser910 and Ser935.
14-3-3 proteins interact dynamically with many intracellular
proteins, which exerts a widespread influence on diverse cellular
processes. They operate by binding to specific phosphorylated
residues on target proteins. The finding that LRRK2 interacts with
14-3-3 isoforms could not be predicted by analysis of the primary
sequence, because the residues surrounding the 910 and 935
phosphorylation sites do not adhere to the optimal Mode 1 and 2
consensus binding motifs for a common mode of 14-3-3 interaction
[11, 15]. However, many proteins that interact with 14-3-3 do so
via diverse non-predictable atypical binding motifs, presumably
because other structural features contribute to the interactions
[11].
[0144] In all cell lines we have investigated (Swiss 3T3 (FIG. 4)
and HEK-293 (FIG. 5)), phosphorylation of LRRK2 at Ser910 and
Ser935 and hence binding to 14-3-3 was reversed by treatment of
cells with the structurally diverse H-1152 and sunitinib LRRK2
inhibitors. We conclude that dephosphorylation results from
inhibition of LRRK2 kinase activity, as H-1152 as well as sunitinib
is ineffective at inducing dephosphorylation of a drug resistant
LRRK2[T2016A] mutant (FIG. 5). Furthermore, H-1152 and sunitinib
are more potent at inducing dephosphorylation of LRRK2[G2019S] than
wild type LRRK2 (FIG. 5), consistent with these drugs inhibiting
LRRK2[G2019S] two to four-fold more potently than the wild type
LRRK2.
[0145] A key question concerns the mechanism by which LRRK2
controls phosphorylation of Ser910 and Ser935. One possibility is
that Ser910 and Ser935 comprise direct LRRK2 autophosphorylation
sites. However, our data suggest that dephosphorylated LRRK2
isolated from H-1152 or sunitinib treated cells is unable to
phosphorylate itself at Ser910/Ser935 following incubation with
magnesium-ATP (FIG. 6). This is consistent with LRRK2 having a
marked preference for phosphorylating threonine residues over
serine residues as demonstrated by our finding that substituting
the phosphorylated Thr residue in an optimal peptide substrate to a
Ser residue, abolished phosphorylation by LRRK2 [8]. Furthermore, a
number of studies aimed at mapping LRRK2 autophosphorylation sites
have not identified Ser910 or Ser935 [16-18]. A global
phosphoproteomic study of a melanoma tumour identified
phosphorylation of LRRK2 at Ser935 as one of 5600 phosphorylation
sites catalogued on 2250 proteins but this was not investigated
further [19].
[0146] There is significant similarity in the sequences surrounding
Ser910 and Ser935 suggesting a single protein kinase may
phosphorylate both of these residues (FIG. 3G). An implication of
our finding is that the Ser910/Ser935 kinase may be stimulated by
LRRK2 and/or the protein phosphatase(s) that acts on these residues
is inhibited by LRRK2. In future work it will be important to
identify the kinase(s) and/or protein phosphatase(s) that act on
Ser910 and Ser935 and to determine whether they are controlled by
LRRK2.
[0147] Our data suggests that phosphorylation of both Ser910 and
Ser935 is required for stable interaction of 14-3-3 with LRRK2 as
binding as mutation of either Ser910 or Ser935 abolishes
interaction 14-3-3 interaction. 14-3-3 molecules form dimers with
each monomer having the ability to interact with a phosphorylated
residue [15]. Thus, a 14-3-3 dimer has the capacity to interact
with two phosphorylated residues. It is possible that one dimer of
14-3-3 interacts with both phosphorylated Ser910 and phosphorylated
Ser935. We also observed that mutation of either Ser910 or Ser935
to an Ala residue induced a significant dephosphorylation the other
residue (FIG. 3E). This could be explained if 14-3-3 binding
protected LRRK2 from becoming dephosphorylated by a protein
phosphatase. Thus abolishing 14-3-3 binding by mutation of either
Ser910 or Ser935 would promote dephosphorylation of the other site.
14-3-3 binding to other targets such as phosphatidylinositol
4-kinase III beta [20] or Cdc25C [21] has been shown to protect
these enzymes from dephosphorylation, presumably by sterically
shielding phosphorylated residues from protein phosphatases.
[0148] 14-3-3 proteins were originally identified over 42 years ago
as acidic proteins that were highly expressed in the brain [31].
Since then 14-3-3 proteins have been implicated in the regulation
of numerous neurological disorders including Parkinson's disease
[26, 27]. For example, 14-3-3 eta binds to parkin, a protein
mutated in autosomal recessive juvenile parkinsonism, and
negatively regulates its E3 ligase activity [22]. 14-3-3 proteins
interact with alpha-synuclein [23] and have been found in Lewy
bodies in brains of patients with Parkinson's disease [24].
Additionally, 14-3-3 theta, epsilon and gamma was recently shown to
suppress the toxic effects of alpha-synuclein overexpression in a
cell based model of neurotoxicity [25]. Our data suggests that
14-3-3 binding to LRRK2 may be relevant to Parkinson's disease as
strikingly 10 out of 41 mutations studied displayed reduced
phosphorylation of Ser910/Ser935 and binding to 14-3-3 isoforms
(FIG. 12).
[0149] Our data suggest that the 14-3-3 interaction does not
control LRRK2 protein kinase activity, as mutation of Ser910 and/or
Ser935 does not influence LRRK2 catalytic activity (FIG. 3D).
Furthermore, treatment of cells with H-1152 or sunitinib induced
dephosphorylation of Ser910 and Ser935 as well as disrupting 14-3-3
binding, but did not affect endogenous LRRK2 kinase activity (FIG.
6). 14-3-3 binding to LRRK2 may impact on its interaction with a
substrate or other regulators or may influence LRRK2 stability or
cellular localisation. Our experiments indicate that 14-3-3 binding
influences the cytoplasmic localisation of LRRK2, as disruption of
14-3-3 binding by mutation of Ser910/Ser935 caused LRRK2 to
accumulate within cytoplasmic pools. Previous work has revealed
that LRRK2 mutants including LRRK2[R1441C] and LRRK2[Y1699C] when
expressed in cells accumulate within cytoplasmic pools [33, 35,
36], a finding we were able to confirm in this study (FIG. 11B or
FIG. 13). These cytoplasmic pools were suggested to comprise
aggregates of misfolded unstable LRRK2 protein [35]. If this were
the case it may suggest that 14-3-3 plays a role in stabilising
LRRK2. Significantly, we found that seven out of eight mutations
that accumulated within cytoplasmic pools displayed reduced
Ser910/Ser935 phosphorylation and binding to 14-3-3 under
conditions in which wild type LRRK2 and most other LRRK2 mutants
analysed bound 14-3-3 and did not accumulate within cytosolic pools
(FIG. 11B). We also validate these findings by demonstrating that
Ser910/Ser935 phosphorylation and 14-3-3 binding is markedly
reduced in three mouse tissues derived from homozygous R1441c
knockin mice that display impaired dopaminergic neurotransmission
[37].
[0150] In FIG. 12 we subdivide the 41 LRRK2 mutations we have
analysed into six groups based on the impact that the mutations
analysed in this study have on protein kinase activity,
Ser910/Ser935 phosphorylation and 14-3-3-binding as well as
cellular localisation. Only three out of the 41 mutations analysed
markedly enhanced LRRK2 protein kinase two to four-fold.
Strikingly, the LRRK2[T2031S] mutation was more active than the
LRRK2[G2019S] mutant, displaying nearly 4-fold higher activity than
wild type LRRK2. The LRRK2[T2031S] mutation has only been reported
in a single Spanish patient with a family history of PD [38].
Interestingly, Thr2031 lies within the T-loop of the LRRK2 kinase
domain, a region where many kinases are activated by
phosphorylation. A recent study has suggested that a catalytically
active fragment of LRRK2 expressed in insect cells
autophosphorylates at several residues including Thr2031 [16],
although we have thus far never been able to observe
phosphorylation of this site in endogenous or overexpressed LRRK2.
We have found that changing Thr2031 to Ala to prevent
phosphorylation led to a similar increase in activity as the T2031S
mutation (RJN data not shown) and mutation to Glu to mimic
phosphorylation inactivates LRRK2 (ND data not shown). Further work
is warranted to understand the role of Thr2031 and how its mutation
to Ser and/or phosphorylation or other covalent modification
impacts on LRRK2 catalytic activity. The LRRK2[R1728H] mutation
displayed 2-fold increased in kinase activity and was identified in
a single patient with a family history of PD [39]. The Arg1728
residue is located outside the kinase domain in the COR domain
(FIG. 9).
[0151] In FIG. 7 we present a model by which phosphorylation of
Ser910 and Ser935 is dependent upon LRRK2 activity and mediates
binding to 14-3-3 isoforms. Phosphorylation of LRRK2 at Ser910 and
Ser935, or 14-3-3 binding, can be deployed as a cell-based readout
to evaluate the relative potency of LRRK2 inhibitors being
developed. This assay can be deployed in cell lines or tissues of
animals or humans treated with LRRK2 inhibitors. For human patients
administered LRRK2 inhibitors in a clinical trial, the
phosphorylation status of LRRK2 at Ser910 and Ser935 in the blood
could be employed as a biomarker of LRRK2 inhibitor efficacy (as
LRRK2 is strongly expressed in blood cells). We believe this to be
the first, simple, cell-based system that can be used to assess the
efficacy of LRRK2 protein kinase inhibitors, based on measuring
phosphorylation of an endogenous LRRK2 target.
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[0224] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of the invention are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contains certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements.
[0225] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0226] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0227] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0228] Specific embodiments disclosed herein may be further limited
in the claims using consisting of or consisting essentially of
language. When used in the claims, whether as filed or added per
amendment, the transition term "consisting of" excludes any
element, step, or ingredient not specified in the claims. The
transition term "consisting essentially of" limits the scope of a
claim to the specified materials or steps and those that do not
materially affect the basic and novel characteristic(s).
Embodiments of the invention so claimed are inherently or expressly
described and enabled herein.
[0229] Furthermore, numerous references have been made to patents
and printed publications throughout this specification. Each of the
above-cited references and printed publications are individually
incorporated herein by reference in their entirety.
[0230] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that may be employed
are within the scope of the invention. Thus, by way of example, but
not of limitation, alternative configurations of the present
invention may be utilized in accordance with the teachings herein.
Accordingly, the present invention is not limited to that precisely
as shown and described.
Sequence CWU 1
1
451246PRTHomo sapiens 1Met Thr Met Asp Lys Ser Glu Leu Val Gln Lys
Ala Lys Leu Ala Glu 1 5 10 15 Gln Ala Glu Arg Tyr Asp Asp Met Ala
Ala Ala Met Lys Ala Val Thr 20 25 30 Glu Gln Gly His Glu Leu Ser
Asn Glu Glu Arg Asn Leu Leu Ser Val 35 40 45 Ala Tyr Lys Asn Val
Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile 50 55 60 Ser Ser Ile
Glu Gln Lys Thr Glu Arg Asn Glu Lys Lys Gln Gln Met 65 70 75 80 Gly
Lys Glu Tyr Arg Glu Lys Ile Glu Ala Glu Leu Gln Asp Ile Cys 85 90
95 Asn Asp Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Pro Asn Ala Thr
100 105 110 Gln Pro Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp
Tyr Phe 115 120 125 Arg Tyr Leu Ser Glu Val Ala Ser Gly Asp Asn Lys
Gln Thr Thr Val 130 135 140 Ser Asn Ser Gln Gln Ala Tyr Gln Glu Ala
Phe Glu Ile Ser Lys Lys 145 150 155 160 Glu Met Gln Pro Thr His Pro
Ile Arg Leu Gly Leu Ala Leu Asn Phe 165 170 175 Ser Val Phe Tyr Tyr
Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser 180 185 190 Leu Ala Lys
Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu 195 200 205 Asn
Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 210 215
220 Asp Asn Leu Thr Leu Trp Thr Ser Glu Asn Gln Gly Asp Glu Gly Asp
225 230 235 240 Ala Gly Glu Gly Glu Asn 245 2246PRTMus musculus
2Met Thr Met Asp Lys Ser Glu Leu Val Gln Lys Ala Lys Leu Ala Glu 1
5 10 15 Gln Ala Glu Arg Tyr Asp Asp Met Ala Ala Ala Met Lys Ala Val
Thr 20 25 30 Glu Gln Gly His Glu Leu Ser Asn Glu Glu Arg Asn Leu
Leu Ser Val 35 40 45 Ala Tyr Lys Asn Val Val Gly Ala Arg Arg Ser
Ser Trp Arg Val Ile 50 55 60 Ser Ser Ile Glu Gln Lys Thr Glu Arg
Asn Glu Lys Lys Gln Gln Met 65 70 75 80 Gly Lys Glu Tyr Arg Glu Lys
Ile Glu Ala Glu Leu Gln Asp Ile Cys 85 90 95 Asn Asp Val Leu Glu
Leu Leu Asp Lys Tyr Leu Ile Leu Asn Ala Thr 100 105 110 Gln Ala Glu
Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe 115 120 125 Arg
Tyr Leu Ser Glu Val Ala Ser Gly Glu Asn Lys Gln Thr Thr Val 130 135
140 Ser Asn Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys
145 150 155 160 Glu Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala
Leu Asn Phe 165 170 175 Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro
Glu Lys Ala Cys Ser 180 185 190 Leu Ala Lys Thr Ala Phe Asp Glu Ala
Ile Ala Glu Leu Asp Thr Leu 195 200 205 Asn Glu Glu Ser Tyr Lys Asp
Ser Thr Leu Ile Met Gln Leu Leu Arg 210 215 220 Asp Asn Leu Thr Leu
Trp Thr Ser Glu Asn Gln Gly Asp Glu Gly Asp 225 230 235 240 Ala Gly
Glu Gly Glu Asn 245 3255PRTHomo sapiens 3Met Asp Asp Arg Glu Asp
Leu Val Tyr Gln Ala Lys Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr
Asp Glu Met Val Glu Ser Met Lys Lys Val Ala Gly 20 25 30 Met Asp
Val Glu Leu Thr Val Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45
Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser 50
55 60 Ser Ile Glu Gln Lys Glu Glu Asn Lys Gly Gly Glu Asp Lys Leu
Lys 65 70 75 80 Met Ile Arg Glu Tyr Arg Gln Met Val Glu Thr Glu Leu
Lys Leu Ile 85 90 95 Cys Cys Asp Ile Leu Asp Val Leu Asp Lys His
Leu Ile Pro Ala Ala 100 105 110 Asn Thr Gly Glu Ser Lys Val Phe Tyr
Tyr Lys Met Lys Gly Asp Tyr 115 120 125 His Arg Tyr Leu Ala Glu Phe
Ala Thr Gly Asn Asp Arg Lys Glu Ala 130 135 140 Ala Glu Asn Ser Leu
Val Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met 145 150 155 160 Thr Glu
Leu Pro Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn 165 170 175
Phe Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Asp Arg Ala Cys 180
185 190 Arg Leu Ala Lys Ala Ala Phe Asp Asp Ala Ile Ala Glu Leu Asp
Thr 195 200 205 Leu Ser Glu Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met
Gln Leu Leu 210 215 220 Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Met
Gln Gly Asp Gly Glu 225 230 235 240 Glu Gln Asn Lys Glu Ala Leu Gln
Asp Val Glu Asp Glu Asn Gln 245 250 255 4255PRTMus musculus 4Met
Asp Asp Arg Glu Asp Leu Val Tyr Gln Ala Lys Leu Ala Glu Gln 1 5 10
15 Ala Glu Arg Tyr Asp Glu Met Val Glu Ser Met Lys Lys Val Ala Gly
20 25 30 Met Asp Val Glu Leu Thr Val Glu Glu Arg Asn Leu Leu Ser
Val Ala 35 40 45 Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp
Arg Ile Ile Ser 50 55 60 Ser Ile Glu Gln Lys Glu Glu Asn Lys Gly
Gly Glu Asp Lys Leu Lys 65 70 75 80 Met Ile Arg Glu Tyr Arg Gln Met
Val Glu Thr Glu Leu Lys Leu Ile 85 90 95 Cys Cys Asp Ile Leu Asp
Val Gln Asp Lys His Leu Ile Pro Ala Ala 100 105 110 Asn Thr Gly Glu
Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr 115 120 125 His Arg
Tyr Leu Ala Glu Phe Ala Thr Gly Asn Asp Arg Lys Glu Ala 130 135 140
Ala Glu Asn Ser Leu Val Ala Tyr Lys Ala Ala Ser Asp Ile Ala Met 145
150 155 160 Thr Glu Leu Pro Pro Thr His Pro Ile Arg Leu Gly Leu Ala
Leu Asn 165 170 175 Phe Ser Val Phe Tyr Tyr Glu Ile Leu Asn Ser Pro
Asp Arg Ala Cys 180 185 190 Arg Leu Ala Lys Ala Ala Phe Asp Asp Ala
Ile Ala Glu Leu Asp Thr 195 200 205 Leu Ser Glu Glu Ser Tyr Lys Asp
Ser Thr Leu Ile Met Gln Leu Leu 210 215 220 Arg Asp Asn Leu Thr Leu
Trp Thr Ser Asp Met Gln Gly Asp Gly Glu 225 230 235 240 Glu Gln Asn
Lys Glu Ala Leu Gln Asp Val Glu Asp Glu Asn Gln 245 250 255
5246PRTHomo sapiens 5Met Gly Asp Arg Glu Gln Leu Leu Gln Arg Ala
Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ser
Ala Met Lys Ala Val Thr Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn
Glu Asp Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Val
Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu
Gln Lys Thr Met Ala Asp Gly Asn Glu Lys Lys Leu Glu 65 70 75 80 Lys
Val Lys Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Thr Val 85 90
95 Cys Asn Asp Val Leu Ser Leu Leu Asp Lys Phe Leu Ile Lys Asn Cys
100 105 110 Asn Asp Phe Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met
Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Ser Gly
Glu Lys Lys Asn 130 135 140 Ser Val Val Glu Ala Ser Glu Ala Ala Tyr
Lys Glu Ala Phe Glu Ile 145 150 155 160 Ser Lys Glu Gln Met Gln Pro
Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175 Leu Asn Phe Ser Val
Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180 185 190 Ala Cys Leu
Leu Ala Lys Gln Ala Phe Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp
Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215
220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Glu
225 230 235 240 Glu Ala Gly Glu Gly Asn 245 6246PRTMus musculus
6Met Gly Asp Arg Glu Gln Leu Leu Gln Arg Ala Arg Leu Ala Glu Gln 1
5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ser Ala Met Lys Ala Val Thr
Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn Glu Asp Arg Asn Leu Leu
Ser Val Ala 35 40 45 Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser
Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu Gln Lys Thr Met Ala Asp
Gly Asn Glu Lys Lys Leu Glu 65 70 75 80 Lys Val Lys Ala Tyr Arg Glu
Lys Ile Glu Lys Glu Leu Glu Thr Val 85 90 95 Cys Asn Asp Val Leu
Ala Leu Leu Asp Lys Phe Leu Ile Lys Asn Cys 100 105 110 Asn Asp Phe
Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly 115 120 125 Asp
Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Ser Gly Glu Lys Lys Asn 130 135
140 Ser Val Val Glu Ala Ser Glu Ala Ala Tyr Lys Glu Ala Phe Glu Ile
145 150 155 160 Ser Lys Glu His Met Gln Pro Thr His Pro Ile Arg Leu
Gly Leu Ala 165 170 175 Leu Asn Phe Ser Val Phe Tyr Tyr Glu Ile Gln
Asn Ala Pro Glu Gln 180 185 190 Ala Cys Leu Leu Ala Lys Gln Ala Phe
Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser
Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn
Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Glu 225 230 235 240 Glu Ala
Gly Glu Gly Asn 245 7247PRTHomo sapiens 7Met Val Asp Arg Glu Gln
Leu Val Gln Lys Ala Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr
Asp Asp Met Ala Ala Ala Met Lys Asn Val Thr Glu 20 25 30 Leu Asn
Glu Pro Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45
Tyr Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50
55 60 Ser Ile Glu Gln Lys Thr Ser Ala Asp Gly Asn Glu Lys Lys Ile
Glu 65 70 75 80 Met Val Arg Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu
Glu Ala Val 85 90 95 Cys Gln Asp Val Leu Ser Leu Leu Asp Asn Tyr
Leu Ile Lys Asn Cys 100 105 110 Ser Glu Thr Gln Tyr Glu Ser Lys Val
Phe Tyr Leu Lys Met Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala
Glu Val Ala Thr Gly Glu Lys Arg Ala 130 135 140 Thr Val Val Glu Ser
Ser Glu Lys Ala Tyr Ser Glu Ala His Glu Ile 145 150 155 160 Ser Lys
Glu His Met Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175
Leu Asn Tyr Ser Val Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180
185 190 Ala Cys His Leu Ala Lys Thr Ala Phe Asp Asp Ala Ile Ala Glu
Leu 195 200 205 Asp Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu
Ile Met Gln 210 215 220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser
Asp Gln Gln Asp Asp 225 230 235 240 Asp Gly Gly Glu Gly Asn Asn 245
8247PRTMus musculus 8Met Val Asp Arg Glu Gln Leu Val Gln Lys Ala
Arg Leu Ala Glu Gln 1 5 10 15 Ala Glu Arg Tyr Asp Asp Met Ala Ala
Ala Met Lys Asn Val Thr Glu 20 25 30 Leu Asn Glu Pro Leu Ser Asn
Glu Glu Arg Asn Leu Leu Ser Val Ala 35 40 45 Tyr Lys Asn Val Val
Gly Ala Arg Arg Ser Ser Trp Arg Val Ile Ser 50 55 60 Ser Ile Glu
Gln Lys Thr Ser Ala Asp Gly Asn Glu Lys Lys Ile Glu 65 70 75 80 Met
Val Arg Ala Tyr Arg Glu Lys Ile Glu Lys Glu Leu Glu Ala Val 85 90
95 Cys Gln Asp Val Leu Ser Leu Leu Asp Asn Tyr Leu Ile Lys Asn Cys
100 105 110 Ser Glu Thr Gln Tyr Glu Ser Lys Val Phe Tyr Leu Lys Met
Lys Gly 115 120 125 Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala Thr Gly
Glu Lys Arg Ala 130 135 140 Thr Val Val Glu Ser Ser Glu Lys Ala Tyr
Ser Glu Ala His Glu Ile 145 150 155 160 Ser Lys Glu His Met Gln Pro
Thr His Pro Ile Arg Leu Gly Leu Ala 165 170 175 Leu Asn Tyr Ser Val
Phe Tyr Tyr Glu Ile Gln Asn Ala Pro Glu Gln 180 185 190 Ala Cys His
Leu Ala Lys Thr Ala Phe Asp Asp Ala Ile Ala Glu Leu 195 200 205 Asp
Thr Leu Asn Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln 210 215
220 Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr Ser Asp Gln Gln Asp Asp
225 230 235 240 Asp Gly Gly Glu Gly Asn Asn 245 9245PRTHomo sapiens
9Met Glu Lys Thr Glu Leu Ile Gln Lys Ala Lys Leu Ala Glu Gln Ala 1
5 10 15 Glu Arg Tyr Asp Asp Met Ala Thr Cys Met Lys Ala Val Thr Glu
Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser
Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Gly Arg Arg Ser Ala Trp
Arg Val Ile Ser Ser 50 55 60 Ile Glu Gln Lys Thr Asp Thr Ser Asp
Lys Lys Leu Gln Leu Ile Lys 65 70 75 80 Asp Tyr Arg Glu Lys Val Glu
Ser Glu Leu Arg Ser Ile Cys Thr Thr 85 90 95 Val Leu Glu Leu Leu
Asp Lys Tyr Leu Ile Ala Asn Ala Thr Asn Pro 100 105 110 Glu Ser Lys
Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Phe Arg Tyr 115 120 125 Leu
Ala Glu Val Ala Cys Gly Asp Asp Arg Lys Gln Thr Ile Asp Asn 130 135
140 Ser Gln Gly Ala Tyr Gln Glu Ala Phe Asp Ile Ser Lys Lys Glu Met
145 150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn
Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Asn Pro Glu Leu
Ala Cys Thr Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala
Glu Leu Asp Thr Leu Asn Glu 195 200 205 Asp Ser Tyr Lys Asp Ser Thr
Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr
Ser Asp Ser Ala Gly Glu Glu Cys Asp Ala Ala 225 230 235 240 Glu Gly
Ala Glu Asn 245 10245PRTMus musculus 10Met Glu Lys Thr Glu Leu Ile
Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp
Met Ala Thr Cys Met Lys Ala Val Thr Glu Gln 20 25 30 Gly Ala Glu
Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45
Lys
Asn Val Val Gly Gly Arg Arg Ser Ala Trp Arg Val Ile Ser Ser 50 55
60 Ile Glu Gln Lys Thr Asp Thr Ser Asp Lys Lys Leu Gln Leu Ile Lys
65 70 75 80 Asp Tyr Arg Glu Lys Val Glu Ser Glu Leu Arg Ser Ile Cys
Thr Thr 85 90 95 Val Leu Glu Leu Leu Asp Lys Tyr Leu Ile Ala Asn
Ala Thr Asn Pro 100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys
Gly Asp Tyr Phe Arg Tyr 115 120 125 Leu Ala Glu Val Ala Cys Gly Asp
Asp Arg Lys Gln Thr Ile Glu Asn 130 135 140 Ser Gln Gly Ala Tyr Gln
Glu Ala Phe Asp Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr
His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe
Tyr Tyr Glu Ile Leu Asn Asn Pro Glu Leu Ala Cys Thr Leu Ala 180 185
190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Asn Glu
195 200 205 Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg
Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Ser Ala Gly Glu Glu
Cys Asp Ala Ala 225 230 235 240 Glu Gly Ala Glu Asn 245
11245PRTHomo sapiens 11Met Asp Lys Asn Glu Leu Val Gln Lys Ala Lys
Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp Met Ala Ala Cys
Met Lys Ser Val Thr Glu Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu
Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly
Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser 50 55 60 Ile Glu Gln
Lys Thr Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg 65 70 75 80 Glu
Tyr Arg Glu Lys Ile Glu Thr Glu Leu Arg Asp Ile Cys Asn Asp 85 90
95 Val Leu Ser Leu Leu Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Ala
100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr
Arg Tyr 115 120 125 Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys Gly
Ile Val Asp Gln 130 135 140 Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu
Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr His Pro Ile Arg
Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile
Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala 180 185 190 Lys Thr Ala
Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu 195 200 205 Glu
Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215
220 Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly
225 230 235 240 Glu Gly Gly Glu Asn 245 12245PRTMus musculus 12Met
Asp Lys Asn Glu Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10
15 Glu Arg Tyr Asp Asp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln
20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val
Ala Tyr 35 40 45 Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg
Val Val Ser Ser 50 55 60 Ile Glu Gln Lys Thr Glu Gly Ala Glu Lys
Lys Gln Gln Met Ala Arg 65 70 75 80 Glu Tyr Arg Glu Lys Ile Glu Thr
Glu Leu Arg Asp Ile Cys Asn Asp 85 90 95 Val Leu Ser Leu Leu Glu
Lys Phe Leu Ile Pro Asn Ala Ser Gln Pro 100 105 110 Glu Ser Lys Val
Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr 115 120 125 Leu Ala
Glu Val Ala Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln 130 135 140
Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met 145
150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe
Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala
Cys Ser Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu
Leu Asp Thr Leu Ser Glu 195 200 205 Glu Ser Tyr Lys Asp Ser Thr Leu
Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser
Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly 225 230 235 240 Glu Gly Gly
Glu Asn 245 13248PRTHomo sapiens 13Met Glu Arg Ala Ser Leu Ile Gln
Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Glu Asp Met
Ala Ala Phe Met Lys Gly Ala Val Glu Lys 20 25 30 Gly Glu Glu Leu
Ser Cys Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn
Val Val Gly Gly Gln Arg Ala Ala Trp Arg Val Leu Ser Ser 50 55 60
Ile Glu Gln Lys Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu 65
70 75 80 Val Arg Glu Tyr Arg Glu Lys Val Glu Thr Glu Leu Gln Gly
Val Cys 85 90 95 Asp Thr Val Leu Gly Leu Leu Asp Ser His Leu Ile
Lys Glu Ala Gly 100 105 110 Asp Ala Glu Ser Arg Val Phe Tyr Leu Lys
Met Lys Gly Asp Tyr Tyr 115 120 125 Arg Tyr Leu Ala Glu Val Ala Thr
Gly Asp Asp Lys Lys Arg Ile Ile 130 135 140 Asp Ser Ala Arg Ser Ala
Tyr Gln Glu Ala Met Asp Ile Ser Lys Lys 145 150 155 160 Glu Met Pro
Pro Thr Asn Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe 165 170 175 Ser
Val Phe His Tyr Glu Ile Ala Asn Ser Pro Glu Glu Ala Ile Ser 180 185
190 Leu Ala Lys Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu
195 200 205 Ser Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu
Leu Arg 210 215 220 Asp Asn Leu Thr Leu Trp Thr Ala Asp Asn Ala Gly
Glu Glu Gly Gly 225 230 235 240 Glu Ala Pro Gln Glu Pro Gln Ser 245
14248PRTMus musculus 14Met Glu Arg Ala Ser Leu Ile Gln Lys Ala Lys
Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Glu Asp Met Ala Ala Phe
Met Lys Ser Ala Val Glu Lys 20 25 30 Gly Glu Glu Leu Ser Cys Glu
Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly
Gly Gln Arg Ala Ala Trp Arg Val Leu Ser Ser 50 55 60 Ile Glu Gln
Lys Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu 65 70 75 80 Val
Lys Glu Tyr Arg Glu Lys Val Glu Thr Glu Leu Arg Gly Val Cys 85 90
95 Asp Thr Val Leu Gly Leu Leu Asp Ser His Leu Ile Lys Gly Ala Gly
100 105 110 Asp Ala Glu Ser Arg Val Phe Tyr Leu Lys Met Lys Gly Asp
Tyr Tyr 115 120 125 Arg Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys
Lys Arg Ile Ile 130 135 140 Asp Ser Ala Arg Ser Ala Tyr Gln Glu Ala
Met Asp Ile Ser Lys Lys 145 150 155 160 Glu Met Pro Pro Thr Asn Pro
Ile Arg Leu Gly Leu Ala Leu Asn Phe 165 170 175 Ser Val Phe His Tyr
Glu Ile Ala Asn Ser Pro Glu Glu Ala Ile Ser 180 185 190 Leu Ala Lys
Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu 195 200 205 Ser
Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 210 215
220 Asp Asn Leu Thr Leu Trp Thr Ala Asp Ser Ala Gly Glu Glu Gly Gly
225 230 235 240 Glu Ala Pro Glu Glu Pro Gln Ser 245 1515PRTHomo
sapiens 15Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gln Ile Arg
Gln 1 5 10 15 1621PRTHomo sapiens 16Arg Leu Gly Arg Asp Lys Tyr Lys
Thr Leu Arg Gln Ile Arg Gln Gly 1 5 10 15 Asn Thr Lys Gln Arg 20
1721PRTHomo sapiens 17Arg Leu Gly Trp Trp Arg Phe Tyr Thr Leu Arg
Arg Ala Arg Gln Gly 1 5 10 15 Asn Thr Lys Gln Arg 20 1814PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION 18Val Lys Lys Lys Ser Asn Ser
Ile Ser Val Gly Glu Phe Tyr 1 5 10 1915PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION 19Asn Leu Gln Arg His Ser Asn
Ser Leu Gly Pro Ile Phe Asp His 1 5 10 15 2022PRTMus
musculusMOD_RES(11)..(11)PHOSPHORYLATION 20Asn Thr Leu Gln Glu Gly
Val Ala Ser Gly Ser Asp Gly Asn Phe Ser 1 5 10 15 Glu Asp Ala Leu
Ala Lys 20 2111PRTMus musculusMOD_RES(3)..(3)PHOSPHORYLATION 21Ser
Asn Ser Ile Ser Val Gly Glu Val Tyr Arg 1 5 10 2212PRTMus
musculusMOD_RES(4)..(4)PHOSPHORYLATION 22Lys Ser Asn Ser Ile Ser
Val Gly Glu Val Tyr Arg 1 5 10 2316PRTMus
musculusMOD_RES(4)..(4)PHOSPHORYLATION 23His Ser Asn Ser Leu Gly
Pro Val Phe Asp His Glu Asp Leu Leu Arg 1 5 10 15 2422PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION 24Ser Ala Val Glu Glu Gly
Thr Ala Ser Gly Ser Asp Gly Asn Phe Ser 1 5 10 15 Glu Asp Val Leu
Ser Lys 20 2511PRTHomo sapiensMOD_RES(3)..(3)PHOSPHORYLATION 25Ser
Asn Ser Ile Ser Val Gly Glu Phe Tyr Arg 1 5 10 2612PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION 26Lys Ser Asn Ser Ile Ser Val
Gly Glu Phe Tyr Arg 1 5 10 2717PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION 27His Ser Asn Ser Leu Gly Pro
Ile Phe Asp His Glu Asp Leu Leu Lys 1 5 10 15 Arg 289PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION 28Ile Leu Ser Ser Asp Asp Ser
Leu Arg 1 5 2912PRTHomo sapiensMOD_RES(4)..(4)PHOSPHORYLATION 29His
Ser Asp Ser Ile Ser Ser Leu Ala Ser Glu Arg 1 5 10 3012PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION 30His Ser Asp Ser Ile Ser Ser
Leu Ala Ser Glu Arg 1 5 10 3137PRTHomo sapiens 31Lys Lys Lys Ser
Asn Ser Ile Ser Val Gly Glu Phe Tyr Arg Asp Ala 1 5 10 15 Val Leu
Gln Arg Cys Ser Pro Asn Leu Gln Arg His Ser Asn Ser Leu 20 25 30
Gly Pro Ile Phe Asp 35 3237PRTMus musculus 32Lys Arg Lys Ser Asn
Ser Ile Ser Val Gly Glu Val Tyr Arg Asp Leu 1 5 10 15 Ala Leu Gln
Arg Tyr Ser Pro Asn Ala Gln Arg His Ser Asn Ser Leu 20 25 30 Gly
Pro Val Phe Asp 35 3337PRTRattus norvegicus 33Lys Lys Lys Ser Asn
Ser Val Ser Val Gly Glu Val Tyr Arg Asp Leu 1 5 10 15 Ala Leu Gln
Arg Cys Ser Pro Asn Ala Gln Arg His Ser Ser Ser Leu 20 25 30 Gly
Pro Val Phe Asp 35 3437PRTBos taurus 34Lys Thr Lys Ser Asn Ser Ile
Ser Val Gly Glu Phe Tyr Gln Asp Pro 1 5 10 15 Ala Leu Gln Arg Cys
Ser Pro Asn Leu Gln Arg His Ser Ser Ser Leu 20 25 30 Gly Pro Ile
Phe Asp 35 3537PRTCanis familiaris 35Lys Arg Lys Ser Asn Ser Ile
Ser Val Gly Glu Phe Tyr His Asp Arg 1 5 10 15 Ala Leu Gln Arg Cys
Ser Pro Asn Leu Gln Arg His Ser Asn Ser Leu 20 25 30 Gly Pro Ile
Phe Asp 35 3638PRTGallus gallus 36Lys Lys Lys Ser Asn Ser Ile Ala
Val Ala Asp Leu His Cys Arg Glu 1 5 10 15 Leu Ala Phe Gln Arg Gly
Ser Pro Thr Leu Pro Arg His Ser Tyr Ser 20 25 30 Val Gly Pro Gly
Ser Asp 35 3713PRTHomo sapiens 37Val Lys Lys Lys Ser Asn Ser Ile
Ser Val Gly Glu Phe 1 5 10 3813PRTHomo sapiens 38Leu Gln Arg His
Ser Asn Ser Leu Gly Pro Ile Phe Asp 1 5 10 3952PRTHomo sapiens
39Asp Ser Glu Gly Ser Glu Gly Ser Phe Leu Val Lys Lys Lys Ser Asn 1
5 10 15 Ser Ile Ser Val Gly Glu Phe Tyr Arg Asp Ala Val Leu Gln Arg
Cys 20 25 30 Ser Pro Asn Leu Gln Arg His Ser Asn Ser Leu Gly Pro
Ile Phe Asp 35 40 45 His Glu Asp Leu 50 4052PRTPan troglodytes
40Asp Ser Glu Gly Ser Glu Gly Ser Phe Leu Val Lys Arg Lys Ser Asn 1
5 10 15 Ser Ile Ser Val Gly Glu Phe Tyr Arg Asp Ala Val Leu Gln Arg
Cys 20 25 30 Ser Pro Asn Leu Gln Arg His Ser Asn Ser Leu Gly Pro
Ile Phe Asp 35 40 45 His Glu Asp Leu 50 4152PRTMus musculus 41Asp
Ser Glu Gly Ser Glu Ser Ser Phe Leu Val Lys Arg Lys Ser Asn 1 5 10
15 Ser Ile Ser Val Gly Glu Val Tyr Arg Asp Leu Ala Leu Gln Arg Tyr
20 25 30 Ser Pro Asn Ala Gln Arg His Ser Asn Ser Leu Gly Pro Val
Phe Asp 35 40 45 His Glu Asp Leu 50 4252PRTRattus norvegicus 42Asp
Ser Glu Gly Ser Glu Ser Ser Phe Leu Val Lys Lys Lys Ser Asn 1 5 10
15 Ser Val Ser Val Gly Glu Val Tyr Arg Asp Leu Ala Leu Gln Arg Cys
20 25 30 Ser Pro Asn Ala Gln Arg His Ser Ser Ser Leu Gly Pro Val
Phe Asp 35 40 45 His Glu Asp Leu 50 4352PRTBos taurus 43Asp Ser Glu
Gly Ser Glu Gly Ser Phe Leu Val Lys Thr Lys Ser Asn 1 5 10 15 Ser
Ile Ser Val Gly Glu Phe Tyr Gln Asp Pro Ala Leu Gln Arg Cys 20 25
30 Ser Pro Asn Leu Gln Arg His Ser Ser Ser Leu Gly Pro Ile Phe Asp
35 40 45 His Glu Asp Leu 50 4452PRTCanis familiaris 44Asp Ser Glu
Gly Ser Glu Gly Ser Phe Leu Val Lys Arg Lys Ser Asn 1 5 10 15 Ser
Ile Ser Val Gly Glu Phe Tyr His Asp Arg Ala Leu Gln Arg Cys 20 25
30 Ser Pro Asn Leu Gln Arg His Ser Asn Ser Leu Gly Pro Ile Phe Asp
35 40 45 His Glu Asp Phe 50 4553PRTGallus gallus 45Asp Ser Glu Gly
Ser Glu Gly Ser Val Phe Arg Lys Lys Lys Ser Asn 1 5 10 15 Ser Ile
Ala Val Ala Asp Leu His Cys Arg Glu Leu Ala Phe Gln Arg 20 25 30
Gly Ser Pro Thr Leu Pro Arg His Ser Tyr Ser Val Gly Pro Gly Ser 35
40 45 Asp Tyr Glu Pro Leu 50
* * * * *
References