Maria
Emanuela Cuomo, Axel Knebel, Nick Morrice, Hugh Paterson,
Philip Cohen and Sibylle Mittnacht
Nature
Cell Biology May 2008 in press
p53-Driven
apoptosis limits centrosome amplification and genomic instability
downstream of NPM1 phosphorylation
Chromosome
loss or gain is associated with a large number of solid cancers,
providing genomic plasticity and thus adaptability to cancer
cells. Numerical centrosome abnormalities arising from centrosome
over-duplication or failed cytokinesis are a recognized cause
of aneuploidy. In higher eukaryotic cells, the centrosome
duplicates only once per cell cycle to ensure the formation
of a bipolar mitotic spindle that orchestrates the balanced
distribution of the sister chromatids to the respective daughter
cells. Here we delineate the events that allow abnormal centrosome
duplication, resulting in mitotic errors and incorrect chromosome
segregation in cells with sustained cyclin-dependent kinase
(CDK) activity. We have identified NPM1 as a substrate for
CDK6 activated by the Kaposi's sarcoma herpesvirus (KSHV)
D-type cyclin and shown that p53-driven apoptosis occurs downstream
of NPM1 phosphorylation as a checkpoint mechanism that prevents
accumulation of cells with supernumerary centrosomes. Our
findings provide evidence that abnormal chromosome segregation
in KSHV-infected cells is a direct consequence of NPM1 phosphorylation
and predict that genomic instability is an inevitable consequence
of latent KSHV infection.
_____________________________________________________
Mahaboobi
Jaleel, R. Jeremy Nichols, Maria Deak, David G. Campbell,
Frank Gillardon, Axel Knebel and Dario R Alessi
J.
Biochem. 2007 405:307-317
LRRK2
phosphorylates moesin at Thr558; characterisation of how Parkinson's
disease mutants affect kinase activity.
Mutations in the Leucine Rich Repeat Kinase-2 (LRRK2) gene
cause late-onset Parkinson's disease (PD). LRRK2, contains
Leucine Rich Repeats, a GTPase domain, a COR domain, a kinase
and a WD40 motif. Little is known about how LRRK2 is regulated,
what its physiological substrates are or how mutations affect
LRRK2 function. Thus far LRRK2 activity has only been assessed
by autophosphorylation and phosphorylation of myelin basic
protein, which is catalysed rather slowly. We undertook a
KESTREL screen in rat brain extracts to identify proteins
that were phosphorylated by an activated PD mutant of LRRK2
(G2019S). This led to the discovery that moesin, a protein
which anchors the actin-cytoskeleton to the plasma membrane,
is efficiently phosphorylated by LRRK2, at Thr558, a previously
identified in vivo phosphorylation site that regulates the
ability of moesin to bind actin. LRRK2 also phosphorylated
ezrin and radixin that are related to moesin, at the residue
equivalent to Thr558, as well as a peptide encompassing Thr558
(LRRKtide). We exploited these findings to determine how nine
previously reported PD mutations of LRRK2 affected kinase
activity. Only one of the mutations analysed, namely G2019S,
stimulated kinase activity. Four mutations inhibited LRRK2
kinase activity (R1941H, I2012T, I2020T and G2385R), whereas
the remainder (R1441C, R1441G, Y1699C and T2356I), did not
influence activity. Therefore, the manner in which LRRK2 mutations
induce PD, is more complex than previously imagined, and not
only caused by an increase of LRRK2 kinase activity. Finally,
we show that the minimum catalytically active fragment of
LRRK2, requires an intact GTPase, COR and kinase domain as
well as WD40 motif and C-terminal tail. This study suggests
that moesin, ezrin and radixin may comprise LRRK2 substrates,
findings that have been exploited to develop the first robust
quantitative assay to measure LRRK2 kinase activity.
_____________________________________________________
Charline
Peng, Axel Knebel, Nick A. Morrice, Xiang J. Li, Kevin Barringer,
Jun Li, Scott Jakes, Brian Werneburg and Lian Wang
Journal
of Biochemistry 141:353-362
Pim
Kinase Substrate Identification and Specificity.
The Pim
family of Ser/Thr kinases has been implicated in the process
of lymphomagenesis and cell survival. Known substrates of
Pim kinases are few and poorly characterized. In this study
we set out to identify novel Pim-2 substrates using the KESTREL
(Kinase Substrate Tracking and Elucidation) approach. Two
potential substrates, eukaryotic initiation factor 4B (eIF4B)
and apoptosis inhibitor 5 (API-5), were identified from rat
thymus extracts. Sequence comparison of the Pim-2 kinase phosphorylation
sites of eIF4B and mouse BAD, the only other known Pim-2 substrate,
revealed conserved amino acids preceding the phosphorylated
serine residue. Stepwise replacement of the conserved residues
produced a consensus sequence for Pim kinases recognition:
RXRHXS. Pim-1 and Pim-2 catalyzed the phosphorylation of this
recognition sequence 20-fold more efficiently than the original
(K/R-K/R-R-K/R-L-S/T-a; a = small chain amino acid) Pim-1
phosphorylation site. The identification of the novel Pim
kinases consensus sequence provides a more sensitive and versatile
peptide based assay for screening modulators of Pim kinases
activity.
_______________________________________________________
Philip
Cohen and Axel Knebel
Biochem.
J. 2006 393:1-6
KESTREL:
a powerful method for identifying the physiological substrates
of protein kinases.[Review]
The identification
of all the substrates of every protein kinase is one of the
major challenges of post-genomic research. Here we review
a powerful method for tackling this problem that we have developed
over the last 5 years. The method has so far been used to
identify novel substrates for eight different protein kinases,
demonstrating that it is of general utility. Importantly,
the method can be used to identify distinct physiological
substrates of protein kinases, such as PKB (protein kinase
B) and SGK (serum- and glucocorticoid-induced kinase), that
are closely related in structure and have similar specificity
determinants.
________________________________________________________
Maria
Emanuela Cuomo, Axel Knebel, Georgina Platt,Nick A. Morrice,
Philip Cohen and Sybille Mittnacht
J.
Biol. Chem. (2005) 280:35844-58
Regulation
of microfilament organization by KSHV- cyclin/ cdk phosphorylation
of caldesmon
Kaposi
Sarcoma- Associated Herpes Virus (KSHV) encodes a D- like
cyclin (K- cyclin) that is thought to contribute to the viral
oncogenicity. K- cyclin activates cellular cyclin dependent
kinase (cdk) 4 and 6, generating enzymes with a substrate
selectivity deviant from cdk4/ 6 activated by D- type cyclins,
suggesting different biochemical and biological functions.
Here we report the identification of the actin- and calmodulin-
binding protein caldesmon (CALD1) as a novel K- cyclin/ cdk
substrate, which is not phosphorylated by D/ cdk. CALD1 plays
a central role in the regulation of microfilament organisation,
consequently controlling cell shape, adhesion, cytokinesis
and motility. K- cyclin/ cdk6 specifically phosphorylates
four S/T sites in the human CALD1 carboxy- terminus, abolishing
CALD1 binding to its effector protein actin and its regulator
protein calmodulin. CALD1 is hyper- phosphorylated in cells
following K- cyclin expression and in KSHV transformed lymphoma
cells. Moreover, expression of exogenous K-cyclin results
in microfilament loss and changes in cell morphology; both
effects are reliant on cdk catalysis and can be reverted by
the expression of a phosphorylation defective CALD1. Together,
these data strongly suggest that K- cyclin expression modulates
the activity of caldesmon and through this microfilament functions
in cells. These results establish a novel link between KSHV
infection and the regulation of the actin cytoskeleton.
_________________________________________________________________
Gillian
C Auld, David G Campbell, Nicholas Morrice and Philip Cohen
Biochem
J. (2005) 389:775-783
Identification
of calcium-regulated heat stable protein of 24 kDa (CRHSP24)
as a physiological substrate for PKB and RSK using KESTREL
A
substrate for PKBa was detected in liver extracts that was
purified and identified as the calcium-regulated heat stable
protein of apparent molecular mass 24 kDa (CRHSP24). PKBa,
as well as SGK1 and RSK, phosphorylated CRHSP24 stoichiometrically
at Ser52 in vitro and its brain-specific isoform PIPPin at
the equivalent residue (Ser58). CRHSP24 became phosphorylated
at Ser52 when human 293 cells were stimulated with IGF-1 and
this was prevented by inhibitors of PtdIns 3-kinase, but not
by rapamycin (an inhibitor of mTOR) or PD 184352, an
inhibitor of the classical MAP kinase cascade and hence the
activation of RSK. IGF-1 induced a similar phosphorylation
of CRHSP24 in ES cells from wild-type mice or mice that express
the PDK1[L155E] mutant that activates PKB normally but cannot
activate SGK. CRHSP24 also became phosphorylated at Ser52
in response to EGF and this was prevented by blocking activation
of both the classical MAP kinase cascade and the activation
of PKB, but not if just one of these pathways was inhibited.
DYRK2 phosphorylated CRHSP24 at Ser30, Ser32 and Ser41 in
vitro and Ser41 was identified as a site phosphorylated in
cells. These and other results demonstrate that CRHSP24 is
phosphorylated at Ser52 by PKB in response to IGF-1, at Ser52
by PKB and RSK in response to EGF, and at Ser41 in the absence
of IGF1/EGF by a DYRK isoform or another proline-directed
protein kinase(s).
_________________________________________________________
Robert
A Cartlidge, Axel Knebel, Mark Peggie, Andrei Alexandrov,
Eric M Phizicky, and Philip Cohen
EMBO
J. (2005) 24:1696-1705
The
tRNA methylase METTL1 is phosphorylated and inactivated by
PKB and RSK in vitro and in cells.
A substrate for protein kinase B (PKB) in HeLa cell extracts
was identified as methyltransferase-like protein-1 (METTL1),
the orthologue of trm8, which catalyses the 7-methylguanosine
modification of tRNA in Saccharomyces cerevisiae. PKB and
ribosomal S6 kinase (RSK) both phosphorylated METTL1 at Ser27
in vitro. Ser27 became phosphorylated when HEK293 cells were
stimulated with insulin-like growth factor-1 (IGF-1) and this
was prevented by inhibition of phosphatidyinositol 3-kinase.
The IGF-1-induced Ser27 phosphorylation did not occur in 3-phosphoinositide-dependent
protein kinase-1 (PDK1)-deficient embryonic stem cells, but
occurred normally in PDK1[L155E] cells, indicating that the
effect of IGF-1 is mediated by PKB. METTL1 also became phosphorylated
at Ser27 in response to phorbol-12-myristate 13-acetate and
this was prevented by PD 184352 or pharmacological inhibition
of RSK. Phosphorylation of METTL1 by PKB or RSK inactivated
METTL1 in vitro, as did mutation of Ser27 to Asp or Glu. Expression
of METTL1[S27D] or METTL1[S27E] did not rescue the growth
phenotype of yeast lacking trm8. In contrast, expression of
METTL1 or METTL1[S27A] partially rescued growth. These results
demonstrate that METTL1 is inactivated by PKB and RSK in cells,
and the potential implications of this finding are discussed.
_________________________________________________________
Claire
E. Eyers, Helen McNeill, Axel Knebel, Nick A. Morrice, Simon
Arthur, Ana Cuenda and Philip Cohen.
Biochem
J. (2005) 389:127-135
The
phosphorylation of CapZ-interacting protein (CapZIP) by stress-activated
protein kinases triggers its dissociation from CapZ.
A protein
expressed in immune cells and muscle was detected in muscle
extracts as a substrate for several stress-activated protein
kinases. It interacted specifically with the F-actin capping
protein CapZ in splenocytes, and was therefore termed CapZIP
(CapZ-interacting protein). Human CapZIP was phosphorylated
at Ser179 and Ser244 by MAPKAP-K2 or MAPKAP-K3 in vitro. Anisomycin
induced the phosphorylation of CapZIP at Ser179 in Jurkat
cells, which was prevented by SB 203580, consistent with phosphorylation
by MAPKAP-K2 and/or MAPKAP-K3. However, osmotic shock-induced
phosphorylation of Ser179 was unaffected by SB 203580. These
and other results suggest that CapZIP is phosphorylated at
Ser179 in cells by MAPKAP-K2/MAPKAP-K3 and at least one other
protein kinase. Stress activated MAP kinase family members
phosphorylated human CapZIP at many sites including Ser68,
Ser83, Ser108 and Ser216. Ser108 became phosphorylated when
Jurkat cells were exposed to osmotic shock, which was unaffected
by SB 203580 and/or PD 184352 or in splenocytes from mice
that do not express either SAPK3/p38g or SAPK4/p38d. Our results
suggest that CapZIP may be phosphorylated by JNK, which phosphorylates
CapZIP to >5 mol/mol within minutes in vitro. Osmotic shock
or anisomycin triggered the dissociation of CapZIP from CapZ
in Jurkat cells, suggesting that phosphorylation of CapZIP
may regulate the ability of CapZ to remodel actin filament
assembly in vivo.
_________________________________________________________
Adam
R. Cole, Axel Knebel, Nick A. Morrice, Laura S. Robertson,
Andrew J. Irving, Chris N. Connolly, and Calum Sutherland
J.
Biol. Chem. (2004) Vol 279 pp50176-50180
GSK-3
phosphorylation of the Alzheimers epitope within collapsin
response mediator proteins regulates axon elongation in primary
neurons.
Elevated Glycogen Synthase Kinase-3 (GSK-3) activity is associated
with Alzheimers disease. We have found that Collapsin Response
Mediator Proteins (CRMP) 2 and 4 are physiological substrates
of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated
epitope first identified in plaques isolated from Alzheimers
brain. Expression of wild type CRMP2 in primary hippocampal
neurons or SH-SY5Y neuroblastoma cells promotes axon elongation.
However, a GSK-3-insensitive CRMP2 mutant has dramatically
reduced ability to promote axon elongation, a similar effect
to pharmacological inhibition of GSK-3. Hence, we propose
that phosphorylation of CRMP proteins by GSK-3 regulates axon
elongation. This work provides a direct connection between
hyperphosphorylation of these residues and elevated GSK-3
activity, both of which are observed in Alzheimer brain.
_________________________________________________________________
Murray
JT, Campbell DG, Peggie M, Mora A, Cohen P.
Biochem
J. 2004 Vol 384 pp489-494
The
identification of Filamin C as a new physiological substrate
of PKBalpha using KESTREL.
We detected a protein in rabbit skeletal muscle extracts that
was phosphorylated rapidly by PKBalpha, but not by SGK1, and
identified it as the cytoskeletal protein Filamin C (FLNc).
PKBalpha phosphorylated FLNc at Ser2213 in vitro, which lies
in an insert not present in the FLNa and FLNb isoforms. Ser2213
became phosphorylated when C2C12 myoblasts were stimulated
with insulin or EGF and phosphorylation was prevented by low
concentrations of wortmannin at which it is a relatively specific
inhibitor of phosphatidylinositol 3-kinase. PD 184352 (an
inhibitor of the classical MAP kinase cascade) and/or rapamycin
(an inhibitor of mTOR) had no effect. Insulin also induced
the phosphorylation of FLNc at Ser2213 in cardiac muscle in
vivo, but not in cardiac muscle that does not express PDK1,
the upstream activator of PKB. These results identify the
muscle-specific isoform FLNc as a new physiological substrate
for PKB.
__________________________________________________________________
Murray
JT, Campbell DG, Morrice N, Auld GC, Shpiro N, Marquez R,
Peggie M, Bain J, Bloomberg GB, Grahammer F, Lang F, Wulff
P, Kuhl D, Cohen P.
Biochem
J. 2004 Vol 384 pp477-488
Exploitation of KESTREL to identify N-myc downstream-regulated
gene family members as physiological substrates for SGK1 and
GSK3.
We detected a protein in rabbit skeletal muscle extracts that
was phosphorylated rapidly by SGK1, but not by PKBalpha, and
identified it as N-myc downstream-regulated protein 2 (NDRG2).
SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in
vitro. All three residues were phosphorylated in skeletal
muscle from wild type mice, but not from mice that do not
express SGK1. SGK1 also phosphorylated the related NDRG1 isoform
at Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332
and Thr348 of NDRG2) as well as Thr356 and Thr366. Thr346,
Thr356 and Thr366 are located within identical decapeptide
sequences GTRSRSHTSE, repeated three times in NDRG1. The region
containing these threonines was phosphorylated in liver, lung,
spleen and skeletal muscle of wild type mice, but not in SGK1-/-
mice. "Knockdown" of SGK1 in HeLa cells using small
interfering RNA also suppressed phosphorylation of the threonines
in the repeat region of NDRG1. The phosphorylation of NDRG1
by SGK1 transformed it into an excellent substrate for GSK3,
which could then phosphorylate Ser342, Ser352 and Ser362 in
the repeat region. Incubation of HeLa cells with the specific
GSK3 inhibitor CT 99021 increased the electrophoretic mobility
of NDRG1 in HeLa cells, demonstrating that this protein is
phosphorylated by GSK3 in cells. Our results identify NDRG1
and NDRG2 as physiological substrates for SGK1 and demonstrate
that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation
by GSK3.
________________________________________________________________
McNeill
H, Knebel A, Arthur JS, Cuenda A, Cohen P.
Biochem
J. (2004) Vol 384 pp391-400
A novel UBA and UBX domain protein that binds polyubiquitin
and VCP and is a substrate for SAPKs.
A widely expressed protein containing UBA and UBX domains
was identified as a substrate of stress-activated protein
kinases (SAPKs). Termed SAPK-substrate-1 (SAKS1), it was phosphorylated
efficiently at Ser 200 in vitro by SAPK3/p38gamma, SAPK4/p38delta
and JNK, but weakly by SAPK2a/p38alpha, SAPK2b/p38beta2 or
ERK2. Ser 200, situated immediately N-terminal to the UBX
domain, became phosphorylated in 293 cells in response to
stressors. Phosphorylation was not prevented by SB 203580
(an inhibitor of SAPK2a/p38alpha and SAPK2b/p38beta2) and/or
PD 184352 (which inhibits the activation of ERK1 and ERK2)
and was similar in fibroblasts lacking both SAPK3/p38gamma
and SAPK4/p38delta or JNK1 and JNK2. SAKS1 bound ubiquitin
tetramers and valosin-containing protein (VCP) in vitro via
the UBA and UBX domains, respectively. The amount of VCP in
cell extracts that bound to immobilised GST-SAKS1 was enhanced
by elevating the level of polyubiquitinated proteins, while
SAKS1 and VCP in extracts were co-immunoprecipitated with
an antibody raised against S5a, a component of the 19S proteasomal
subunit that binds polyubiquitinated proteins. Peptide N-glycanase
(PNGase) formed a 1:1 complex with VCP and, for this reason,
also bound to immobilised GST-SAKS1. We suggest that SAKS1
may be an adaptor that directs VCP to polyubiquitinated proteins,
and PNGase to misfolded glycoproteins, facilitating their
destruction by the proteasome.
_________________________________________________________________
Knebel
A, Morrice N, Cohen P.
A
novel method to identify protein kinase substrates: eEF2 kinase
is phosphorylated and inhibited by SAPK4/p38delta.
EMBO
J. 2001 Aug 15;20(16):4360-9
We have developed a method of general application for identifying
putative substrates of protein kinases in cell extracts. Using
this procedure, we identified the physiological substrates
of several mitogen-activated protein kinase kinases and an
authentic substrate of stress-activated protein kinase (SAPK)
2a/p38. A 120 kDa protein was detected in skeletal muscle
extracts that was phosphorylated rapidly by SAPK4/p38delta,
but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular
signal-regulated kinase 2 (ERK2). It was purified and identified
as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta
phosphorylated eEF2K at Ser359 in vitro, causing its inactivation.
eEF2K became phosphorylated at Ser359 and its substrate eEF2
became dephosphorylated (activated) when KB cells were exposed
to anisomycin, an agonist that activates all SAPKs, including
SAPK4/p38delta. The anisomycin-induced phosphorylation of
Ser359 was unaffected by SB 203580, U0126 or rapamycin, and
was prevented by overexpression of a catalytically inactive
SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may
mediate the inhibition of eEF2K by this stress. The phosphorylation
of eEF2K at Ser359 was also induced by insulin-like growth
factor-1. However, this was blocked by rapamycin, indicating
that Ser359 is targeted by at least two signalling pathways.
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