We have cloned the Xenopus laevis homologue (XCL100) of the human CL100 (Thr/Tyr) MAP kinase phosphatase. Expression of the XCL100 mRNA and protein is inducible by serum stimulation and oxidative/heat stress in a X. laevis kidney cell line. In contrast, XCL100 is constitutively expressed in growing Xenopus oocytes. Recombinant XCL100 protein is able to dephosphorylate both tyrosine and threonine residues of activated p42 MAP kinase in vitro and both the Xenopus and human CL100 proteins were localised predominantly in the nucleus in transfected COS-1 cells. As nuclear translocation of activated MAP kinase is necessary for some of its essential functions in proliferation and cell differentiation our results indicate a role for CL100 in the regulation of these nuclear signalling events. In Xenopus kidney cells both heat shock and serum stimulation lead to transient activation of MAP kinase. However, in contrast to results previously reported from studies on mammalian fibroblasts the inactivation of MAP kinase in these epitheloid cells is rapid and is not dependent on synthesis of new protein. These results indicate that the induction of CL100 (or CL100-like enzymes) may not be required for MAP kinase inactivation in all cell types. Finally, during early embryogenesis, levels of XCL100 mRNA are greatly increased at the mid-blastula transition, suggesting that this enzyme may be involved in the regulation of MAP kinase activity during early development.

REFERENCES

Alessi
D. R.
,
Smythe
C.
,
Keyse
S. M.
(
1993
).
The human CL100 gene encodes a tyr/thr-protein phosphatase which potently and specifically inactivates MAP kinase and suppresses its activation by oncogenic ras in Xenopus oocyte extracts.
Oncogene
8
,
2015
2020
Alessi
D. R.
,
Saito
Y.
,
Campbell
D. G.
,
Cohen
P.
,
Sithanandam
G.
,
Rapp
U.
,
Ashworth
A.
,
Marshall
C. J.
,
Cowley
S.
(
1994
).
Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1.
EMBO J
13
,
1610
1619
Alessi
D. R.
,
Gomez
N.
,
Moorhead
G.
,
Lewis
T.
,
Keyse
S. M.
,
Cohen
P.
(
1995
).
Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines.
Curr. Biol
5
,
283
295
Anderson
N. G.
,
Maller
J. L.
,
Tonks
N. K.
,
Sturgill
T. W.
(
1990
).
Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase.
Nature
343
,
651
653
Biggs
W. H.
III
,
Zavitz
K. H.
,
Dickson
B.
,
Van der Straten
A.
,
Brunner
D.
,
Hafen
E.
,
Zipursky
S. L.
(
1994
).
The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.
EMBO J
13
,
1628
1635
Blenis
J.
(
1993
).
Signal transduction via the MAP kinases: proceed at your own RSK.
Proc. Nat. Acad. Sci. USA
90
,
5889
5892
Charles
C. H.
,
Abler
A. S.
,
Lau
L. F.
(
1992
).
cDNA sequence of a growth factor inducible immediate early gene and characterisation of its encoded protein.
Oncogene
7
,
187
190
Charles
C. H.
,
Sun
H.
,
Lau
L. F.
,
Tonks
N. K.
(
1993
).
The growth factor inducible immediate early gene 3CH134 encodes a protein tyrosine phosphatase.
Proc. Nat. Acad. Sci. USA
90
,
5292
5296
Chen
R. H.
,
Sarnecki
C.
,
Blenis
J.
(
1992
).
Nuclear localisation and regulation of erk-and rsk-encoded protein kinases.
Mol. Cell. Biol
12
,
915
927
Chomczynski
P.
,
Saachi
N.
(
1987
).
Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal. Biochem
162
,
156
159
Cobb
M. H.
,
Hepler
J. E.
,
Cheng
M.
,
Robbins
D.
(
1994
).
The mitogen-activated protein-kinases, ERK1 and ERK2.
Semin. Cancer Biol
5
,
261
268
Davis
R. J.
(
1993
).
The mitogen-activated protein-kinase signal transduction pathway.
J. Biol. Chem
268
,
14553
14556
Emslie
E. A.
,
Jones
T. A.
,
Sheer
D.
,
Keyse
S. M.
(
1994
).
The CL100 gene, which encodes a dual-specificity (tyr/thr) MAP kinase phosphatase, is highly conserved and maps to human chromosome 5q34.
Human Genet
93
,
513
516
Feinberg
A. P.
,
Vogelstein
B.
(
1984
).
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activities.
Anal. Biochem
137
,
266
267
Ferrell
J. E.
Jr.
,
Wu
M.
,
Gerhart
J. C.
,
Martin
G. S.
(
1991
).
Cell cycle tyrosine phosphorylation of p34cdc2and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.
Mol. Cell Biol
11
,
1965
1971
Fischer
E. H.
,
Charbonneau
H.
,
Tonks
N. K.
(
1991
).
Protein tyrosine phosphatases: A diverse family of intracellular and transmembrane enzymes.
Science
253
,
401
406
Furuno
N.
,
Nishizawa
M.
,
Okazaki
K.
,
Tanaka
H.
,
Iwashita
J.
,
Nakajo
N.
,
Ogawa
Y.
,
Sagata
N.
(
1994
).
Suppression of DNAreplication via Mos function during meiotic divisions of Xenopus oocytes.
EMBO J
13
,
2399
2410
Gotoh
Y.
,
Nishida
E.
,
Matsuda
S.
,
Shiina
N.
,
Kosako
H.
,
Shiokawa.
K.
,
Akiyama
T.
,
Ohta
K.
,
Sakai
H.
(
1991
).
In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase.
Nature
349
,
251
254
Haccard
O.
,
Sarcevic
B.
,
Lewellyn
A.
,
Hartley
R.
,
Roy
L.
,
Izumi
T.
,
Erikson
E.
,
Maller
J. L.
(
1993
).
Induction of metaphase arrest in cleaving Xenpous embryos by MAP kinase.
Science
262
,
1262
1265
Hoffman
M.
(
1993
).
The cell's nucleus shapes up.
Science
259
,
1257
1259
Ishibashi
T.
,
Bottaro
D. P.
,
Michieli
P.
,
Kelley
C. A.
,
Aaronson
S. A.
(
1994
).
A novel dual specificity phosphatase induced by serum stimulation and heat shock.
J. Biol. Chem
47
,
29897
29902
Kahan
C.
,
Seuwen
K.
,
Meloche
S.
,
Pouyssegur
J.
(
1992
).
Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylyl cyclase inhibition.
J. Biol. Chem
267
,
13369
13375
Kameshita
I.
,
Fujisawa
H.
(
1989
).
A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gels.
Anal. Biochem
183
,
139
143
Kamps
M. P.
,
Sefton
B. M.
(
1989
).
Acid and base hydrolysis of phosphoproteins bound to Immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins.
Anal. Biochem
176
,
22
27
Keyse
S. M.
(
1995
).
An emerging family of dual-specificity MAP kinase phosphatases.
Biochim. Biophys. Acta
1265
,
152
160
Keyse
S. M.
,
Emslie
E. A.
(
1992
).
Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase.
Nature
359
,
644
647
Keyse
S. M.
,
Ginsburg
M.
(
1993
).
Amino-acid-sequence similarity between CL100, a dual-specificity MAP kinase phosphatase and cdc25.
Trends Biochem. Sci
18
,
377
378
Kosako
H.
,
Gotoh
Y.
,
Matsuda
S.
,
Ishikawa
M.
,
Nishida
E.
(
1992
).
Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation.
EMBO J
11
,
2903
2908
Kosako
H.
,
Gotoh
Y.
,
Nishida
E.
(
1994
).
Requirement for the MAP kinase kinase/MAP kinase cascade in Xenopus oocyte maturation.
EMBO J
13
,
2131
2138
Kwak
S. P.
,
Hakes
D. J.
,
Martell
K. J.
,
Dixon
J. E.
(
1994
).
Isolation and characterisation of a human dual specificity protein tyrosine phosphatase gene.
J. Biol. Chem
269
,
3596
3604
Lackner
M. R.
,
Kornfeld
K.
,
Miller
L. M.
,
Horvitz
H. R.
,
Kim
S. K.
(
1994
).
A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans.
Genes Dev
8
,
160
173
Lenormand
P.
,
Sardet
C.
,
Pages
G.
,
L'Allemain
G.
,
Brunet
A.
,
Pouyssegur
J.
(
1993
).
Growth factors induce nuclear translocation of MAP kinases (p42(mapk) and p44(mapk)) but not their activator MAP kinase kinase (p45(mapkk)) in fibroblasts.
J. Cell Biol
122
,
1079
1088
Masui
Y.
,
Clarke
A. J.
(
1979
).
Oocyte maturation.
Int. Rev. Cytol
57
,
185
282
Marshall
C. J.
(
1994
).
MAP kinase kinase kinase, MAP kinase kinase and MAP kinase.
Curr. Opin. Genet. Dev
4
,
82
89
McStay
B.
,
Hu
C. H.
,
Pikaard
C. S.
,
Reeder
R. H.
(
1991
).
xUBF and Rib1 are both required for formation of a stable polymerase I promoter complex in X. laevis.
EMBO J
10
,
2297
2303
Minshull
J.
,
Sun
H.
,
Tonks
N. K.
,
Murray
A. W.
(
1994
).
A MAP kinase dependent spindle assembly checkpoint in Xenopus egg extracts.
Cell
79
,
475
486
Nakielny
S.
,
Cohen
P.
,
Wu
J.
,
Sturgill
T. W.
(
1992
).
MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.
EMBO J
11
,
2123
2129
Nebreda
A. R.
,
Hunt
T.
(
1993
).
The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.
EMBO J
12
,
1979
1986
Nishida
E.
,
Gotoh
Y.
(
1993
).
The MAP kinase cascade is essential for diverse signal transduction pathways.
Trends Biochem. Sci
18
,
128
131
Noguchi
T.
,
Metz
R.
,
Chen
L.
,
Mattei.
M. G.
,
Carrasco
D.
,
Bravo
R.
(
1993
).
Structure, mapping and expression of erp, a growth factor inducible gene encoding a nontransmembrane protein tyrosine phosphatase and effect of ERP on growth.
Mol. Cell. Biol
13
,
5195
5205
Piechaczyk
M.
,
Blanchard
J.
,
Marty
L.
,
Dani
C.
,
Fanabieres
F.
,
El Sabouty
S.
,
Fort
Ph
,
Jeanteur
Ph
(
1984
).
Post-transcriptional regulation of glyceraldehyde 3-phosphate dehydrogenase gene expression in rat tissues.
Nucl. Acids Res
12
,
6951
6963
Posada
J.
,
Yew
N.
,
Ahn
N. G.
,
Vande Woude
G. F.
,
Cooper
J. A.
(
1993
).
Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.
Mol. Cell. Biol
13
,
2546
2553
Roberts
T. M.
(
1992
).
A signal chain of events.
Nature
360
,
534
535
Rohan
P. J.
,
Davis
P.
,
Moskaluk
C. A.
,
Kearns
M.
,
Krutzsch
H.
,
Siebenlist
U.
,
Kelly
K.
(
1993
).
PAC-1: A mitogen-induced nuclear protein tyrosine phosphatase.
Science
259
,
1763
1766
Rouse
J.
,
Cohen
P.
,
Trigon
S.
,
Morange
M.
,
Alonzo-Llamazares
A.
,
Zamanillo
D.
,
Hunt
T.
,
Nebreda
A. R.
(
1994
).
A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.
Cell
78
,
1027
1037
Sagata
N.
,
Oskarsson
M.
,
Copeland
T.
,
Brumbaugh
J.
,
Vande Woude
G. F.
(
1988
).
Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes.
Nature
335
,
519
525
Sanger
F.
,
Nicklen
S.
,
Coulson
A. R.
(
1977
).
DNA sequencing with chain terminating inhibitors.
Proc. Nat. Acad. Sci. USA
74
,
5463
5467
Sarcevic
B.
,
Erikson
E.
,
Maller
J. L.
(
1993
).
Purification and characterisation of a mitogen-activated protein kinase tyrosine phosphatase from Xenopus eggs.
J. Biol. Chem
268
,
25075
25083
Seth
A.
,
Gonzalas
F. A.
,
Gupta
S.
,
Raden
D. L.
,
Davis
R. J.
(
1992
).
Signal transduction within the nucleus by mitogen activated protein kinase.
J. Biol. Chem
267
,
24796
24804
Shaw
G.
,
Kamen
R.
(
1986
).
A conserved AU sequence from the 3untranslated region of GM-CSF mRNA mediates selective mRNA degradation.
Cell
46
,
659
667
Shibuya
E. K.
,
Ruderman
J. V.
(
1993
).
Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells.
Mol. Biol. Cell
4
,
781
790
Sun
H.
,
Charles
C. H.
,
Lau
L. F.
,
Tonks
N. K.
(
1993
).
MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase In vivo.
Cell
75
,
487
493
Traverse
S.
,
Gomez
N.
,
Paterson
H.
,
Marshall
C.
,
Cohen
P.
(
1992
).
Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells.
Biochem. J
288
,
351
355
Ward
Y.
,
Gupta
S.
,
Jensen
P.
,
Wartmann
M.
,
Davis
R. J.
,
Kelly
K.
(
1994
).
Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1.
Nature
367
,
651
654
Wu
J.
,
Lau
L. F.
,
Sturgill
T. W.
(
1994
).
Rapid deactivation of MAP kinase in PC12 cells occurs independently of induction of phosphatase MKP-1.
FEBS Lett
353
,
9
12
Zheng
C. F.
,
Guan
K. L.
(
1993
).
Dephosphorylation and inactivation of the mitogen-activated protein kinase by a mitogen induced Thr/Tyr protein phosphatase.
J. Biol. Chem
268
,
16116
16119
Zheng
C. F.
,
Guan
K. L.
(
1994
).
Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr resides.
EMBO. J
13
,
1123
1131
This content is only available via PDF.