| KESTREL
paper frenzy.
Three
years after the first "KESTREL" paper was published
the ban is broken and four more papers were accepted within
the last 4 weeks, in fact 3 of them within one day. Axel Knebel
explains: "The rate limiting step with KESTREL is not
to find new substrates or even to validate them. What really
takes time is to find out what the substrates actually do
in the cell and then whether the phosphorylation influences
their function. As with the first paper on the regulation
of eEF2 kinase these new findings are also 'door openers'
and have initiated new projects. Numerous substrates are still
being investigated and we can look forward to more exciting
insight into substrate phosphorylation."
Abstracts:
Adam
R. Cole, Axel Knebel, Nick A. Morrice, Laura S. Robertson,
Andrew J. Irving, Chris N. Connolly, and Calum Sutherland
J.
Biol. Chem. 2004 in press
GSK-3
phosphorylation of the Alzheimers epitope within collapsin
response mediator proteins regulates axon elongation in primary
neurons.
Division of Pathology and Neurosciences, University of Dundee,
Dundee, Tayside DD1 9SY, Kinasource Ltd, Laboratory 4.21,
MSI/WTB complex, Dow Street, DD1 5EH, Scotland.
Corresponding Author: c.d.sutherland@dundee.ac.uk
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 Oct 4 [Epub ahead of print]
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 Oct 4 [Epub ahead of print]
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 Sep 13; Pt [Epub ahead of print]
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.
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