Human Myt1 Is a Cell Cycle-regulated Kinase That Inhibits Cdc2 but Not Cdk2 Activity

Activation of the Cdc2·cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. In this study, we have examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. We find that human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreased during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2·cyclin B1 was capable of phosphorylating Myt1 in vitro, but this phosphorylation did not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2·cyclin complexes. Activation of the Cdc2·cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. In this study, we have examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. We find that human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreased during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2·cyclin B1 was capable of phosphorylating Myt1 in vitro, but this phosphorylation did not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2·cyclin complexes. The cyclin-dependent kinases (Cdks) 1The abbreviations used are: Cdk, cyclin-dependent kinase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol. are a family of highly conserved serine/threonine kinases that mediate many of the cell cycle transitions that occur during duplication. Each of these Cdk catalytic subunits associates with a specific subset of regulatory subunits, termed cyclins, to produce a distinct Cdk·cyclin kinase complex that, in general, functions to execute a specific cell cycle event (1Nigg E.A. Bioessays. 1995; 17: 471-480Crossref PubMed Scopus (803) Google Scholar). The placement of a specific Cdk·cyclin complex function with a particular cell cycle transition has led to the identification of key downstream substrates as well as upstream regulatory mechanisms. Well characterized Cdk·cyclin-regulated cell cycle transitions that have been identified thus far include Cdk4·cyclin D and Cdk6·cyclin D complexes with the pRB-gated G1/S transition, Cdk2·cyclin E and Cdk2·cyclin A complexes with the initiation and progression of DNA replication and Cdc2·cyclin A and Cdc2·cyclin B complexes with the initiation of mitosis (reviewed in Refs. 2Nasmyth K. Science. 1996; 274: 1643-1645Crossref PubMed Scopus (327) Google Scholar, 3Stillman B. Science. 1996; 274: 1659-1664Crossref PubMed Scopus (436) Google Scholar, 4Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5029) Google Scholar). Activation of the Cdk·cyclin kinases during these transitions is controlled by a variety of regulatory mechanisms. For the Cdc2·cyclin B complex, inhibition of kinase activity during S phase and G2 is accomplished by phosphorylation of Cdc2 residues Thr14 and Tyr15, which are positioned within the ATP-binding cleft (5Gould K.L. Nurse P. Nature. 1989; 342: 39-45Crossref PubMed Scopus (953) Google Scholar, 6Krek W. Nigg E.A. EMBO J. 1991; 10: 3331-3341Crossref PubMed Scopus (266) Google Scholar, 7Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (253) Google Scholar). Phosphorylation of Thr14and/or Tyr15 is believed to suppress catalytic activity by disrupting the orientation of the ATP molecule present within this cleft (8Atherton-Fessler S. Parker L.L. Geahlen R.L. Piwnica-Worms H. Mol. Cell. Biol. 1993; 13: 1675-1685Crossref PubMed Scopus (153) Google Scholar, 9De Bondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.-H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (844) Google Scholar). The abrupt dephosphorylation of these residues by the Cdc25 phosphatase results in the rapid activation of Cdc2·cyclin B kinase activity and downstream mitotic events (10Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (255) Google Scholar). While phosphorylation/dephosphorylation of the conserved Tyr15site in Cdk2 likely plays an important role in regulating the G1/S transition, the role that Cdk2 Thr14phosphorylation plays is less clear (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar, 12Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A.. 1993; 90: 3521-3524Crossref PubMed Scopus (215) Google Scholar). Phosphorylation of the corresponding inhibitory tyrosine residue in Cdk4 has also been observed (13Terada Y. Tatsuka M. Jinno S. Okayama H. Nature. 1995; 376: 358-362Crossref PubMed Scopus (181) Google Scholar, 14Iavarone A. Massague J. Nature. 1997; 387: 417-421Crossref PubMed Scopus (336) Google Scholar). It has been proposed that Thr14/Tyr15 phosphorylation functions to permit a cell to attain a critical concentration of inactive Cdk·cyclin complexes, which, upon activation, induces a rapid and complete cell cycle transition (15Solomon M.J. Glotzer M. Lee T.H. Philippe M. Kirschner M.W. Cell. 1990; 63: 1013-1024Abstract Full Text PDF PubMed Scopus (560) Google Scholar). There is evidence in mammalian cells that Thr14/Tyr15 phosphorylation also functions, in part, to delay Cdk activation after DNA damage (7Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (253) Google Scholar, 13Terada Y. Tatsuka M. Jinno S. Okayama H. Nature. 1995; 376: 358-362Crossref PubMed Scopus (181) Google Scholar, 16Lock R.B. Ross W.E. Cancer Res. 1990; 50: 3761-3766PubMed Google Scholar, 17Poon R.Y.C. Jiang W. Toyoshima H. Hunter T. J. Biol. Chem. 1996; 271: 13283-13291Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 18Wang Q. Fan S.F. Eastman A. Worland P.J. Sausville E.A. O'Connor P.M. J. Natl. Cancer Inst. 1996; 88: 956-965Crossref PubMed Scopus (454) Google Scholar). The Schizosaccharomyces pombe wee1 gene product was the first kinase identified that is capable of phosphorylating Tyr15 in Cdc2 (19Parker L.L. Atherton-Fessler S. Lee M.S. Ogg S. Falk J.L. Swenson K.I. Piwnica-Worms H. EMBO J. 1991; 10: 1255-1263Crossref PubMed Scopus (164) Google Scholar). Homologs of the Wee1 kinase have been subsequently identified and biochemically characterized from a wide range of species including human, mouse, frog, Saccharomyces cerevisiae, and Drosophila (20Igarashi M. Nagata A. Jinno S. Suto K. Okayama H. Nature. 1991; 353: 80-83Crossref PubMed Scopus (157) Google Scholar, 21Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (559) Google Scholar, 22Watanabe N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Scholar, 23Honda R. Tanaka H. Ohba Y. Yasuda H. Chromosome Res. 1995; 3: 300-308Crossref PubMed Scopus (23) Google Scholar, 24Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (270) Google Scholar, 25Booher R.N. Deshaies R.J. Kirschner M.W. EMBO J. 1993; 12: 3417-3426Crossref PubMed Scopus (290) Google Scholar, 26Campbell S.D. Sprenger F. Edgar B.A. O'Farrell P.H. Mol. Biol. Cell. 1995; 6: 1333-1347Crossref PubMed Scopus (55) Google Scholar). In vertebrate systems, where Thr14 in Cdc2 is also phosphorylated, the Wee1 kinase was capable of phosphorylating Cdc2 on Tyr15, but not Thr14, indicating that another kinase was responsible for Thr14 phosphorylation (21Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (559) Google Scholar, 27McGowan C.H. Russell P. EMBO J. 1993; 12: 75-85Crossref PubMed Scopus (398) Google Scholar). Direct evidence for the existence of a kinase activity that phosphorylated Cdc2 on Thr14 in the membrane fractions ofXenopus egg extracts has been reported (28Kornbluth S. Sebastian B. Hunter T. Newport J. Mol. Biol. Cell. 1994; 5: 273-282Crossref PubMed Scopus (102) Google Scholar). TheXenopus gene encoding this membrane-associated kinase, the Myt1 kinase, has been isolated, and its gene product was shown to be capable of phosphorylating Thr14 and, to a lessor extent, Tyr15 in Cdc2 (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar). A human Myt1 homolog displaying similar properties has been recently identified (30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar) (see “Results”). An apparently unrelated, non-membrane-associated Thr14 kinase activity has also been identified in bovine thymus cytosol (31Matsuura I. Wang J.H. J. Biol. Chem. 1996; 271: 5443-5450Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). While Xenopus Myt1 clearly plays an important role in regulating the rapid early embryonic cell cycles, its role in cells that have distinct G1 and G2 phases as well as growth and checkpoint controls is not known. To investigate the role of Cdk Thr14 phosphorylation in somatic cell cycle control, we have initiated a biochemical analysis of the human Myt1 kinase. The results presented reveal that Myt1, unlike Wee1, exhibits a restricted substrate specificity; it was capable of phosphorylating Thr14 only on Cdc2·cyclin and not Cdk2·cyclin complexes. We also found that endogenous Myt1 kinase activity was reduced during a drug-induced M-phase arrest, concomitant with hyperphosphorylation of the Myt1 protein, implying that Myt1 may be negatively regulated by phosphorylation. A 325-base pair DNA fragment, corresponding to an internal region of EST clones 335241 and 336264, was polymerase chain reaction-generated using oligos 5′-AGCAGCCTCTCCAGCAACTGG-3′ and 5′-CAGAGAAGACCATGGAGTTCC-3′ (5′ and 3′ primers, respectively) and the first strand cDNA synthesis of fetal brain RNA (Clonetech) as a template. This DNA fragment was32P-labeled by random priming (Pharmacia) and used as a probe to screen approximately 7 × 105 clones of a human placental Lambda ZAPII cDNA library (Stratagene). Of nine positives isolated, clone 16 was found to contain the longest cDNA insert. The complete DNA sequence of this 1.98-kilobase pair insert was determined using an ABI sequencer. Frozen pellets of Sf9 cells containing baculovirus-expressed proteins were thawed and lysed by Dounce homogenization in 10 DTT, and by for in a The were and of Cdk, Wee1, and Myt1 were with an ATP × 10 10 for To the Cdk·cyclin kinase activity in these of the was with of a kinase 10 DTT, of for This was with by and by The of Myt1, Wee1 encoding N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google by and were the that the an B. B. I. S. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The Cdc2 in a and the cyclin B1 with in A an Cdc2 and Cdk2 subunits used in this a and have been (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar, Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: PubMed Scopus Google Scholar). The region of which to the (20Igarashi M. Nagata A. Jinno S. Suto K. Okayama H. Nature. 1991; 353: 80-83Crossref PubMed Scopus (157) Google was polymerase chain reaction-generated using the Wee1 clone as a template. Cdk·cyclin complexes were by and Cdk and cyclin to in the of Cdk4·cyclin which was by Sf9 cells with Cdk4 and cyclin Sf9 containing kinase and cyclin Morgan D.O. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar) been to and cyclin B1 during the activation proteins were from Sf9 cell using B. B. I. S. P. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) or using phosphorylation of Cdk·cyclin complexes by Myt1 or kinases were 10 or in with Cdc2·cyclin B1 B1 or B1 complexes in an in kinase containing The was to and by The of the Cdc2 in the Cdc2·cyclin B1 kinase is but a similar has been reported by N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Cdk2·cyclin A Cdk2·cyclin E and Cdk4·cyclin 7 and complexes were or in with Myt1 or in kinase and as in A. cyclin-dependent phosphorylation of Cdc2 by Cdc2·cyclin B1 complexes Cdc2 Cdc2 cyclin B1 or cyclin B1 10 and proteins were or in with Myt1 or in an in kinase and as in A. The in may an endogenous Cdk that with the human cyclin B1 In were by of Cdk·cyclin complexes in of 10 10 containing ATP for by of or Myt1 in of containing of and for The was with and by cell and used in this were from the DNA were a of mammalian that a sequence that the Myt1 proteins an The Myt1 proteins are as residues residues residues containing a to that was by residues containing a of the N. P. S. and A. (30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). The and fractions were from cell that been lysed by Dounce homogenization in an This was for in a to the The membrane-bound proteins in the fractions were by this in the containing for The was by for in a of cells by was by cells for in the of or cells were with Myt1 residues residues and residues were to the and in the and Myt1 were by with Myt1 was used to cells were lysed in with and by × for 15 Myt1 was by of in of containing of for by of of the were with and with To kinase 10 of Cdc2·cyclin B1 kinase of Cdc2·cyclin B1 Sf9 in of containing was to the and for with 10 concentration of the corresponding was during kinase of This was for kinase activity by of a kinase 10 DTT, of of and for 15 with This was with by and by For the were was determined by The of kinase on or of the was mammalian cell and Sf9 cell were by and to using a cell The were with in and 10 with and with and or in was using an Analysis of Myt1 proteins in Xenopus and mitotic egg extracts was as by J. Kirschner M.W. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) that the Myt1 proteins were from Myt1 in a in the of To a human homolog of the Xenopus we a of the EST using the sequence as the (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar). the of were to the of residues within the catalytic of kinases Hunter T. Science. PubMed Scopus Google Scholar). human and were found to within the residues of the kinase. A cDNA clone was and found to the recently human Myt1 kinase (30Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). To human Myt1 we examined its to Cdk·cyclin complexes in a We used the to produce cell extracts that of human Cdk, Wee1, or Myt1 It has been shown that Cdk·cyclin kinase activation be upon Cdk and Gu Y. Morgan D.O. Mol. Biol. Cell. 1992; 3: PubMed Scopus Google of an inhibitory activity be to this We first the of this to Wee1 activity. containing Cdc2 were with cyclin B1 and with or a an of this was and Cdc2·cyclin B1 kinase activity was determined by a kinase shown in of Wee1 Cdc2·cyclin B1 kinase The Wee1 inhibitory activity was on the of the Tyr15 residue in with and and Tyr15 with and proteins to cyclin B1 activity in the of Wee1 Myt1 were with Cdc2 and cyclin a in Cdc2 kinase activation was also observed Myt1 and activation, indicating that Myt1 Thr14 or in it that Myt1 phosphorylates Thr14 kinase activity. To Myt1 Cdk2·cyclin complexes, we the kinase activity of Cdk2 that been in the of We used the as a to for inhibitory activity that may be present in the Myt1 shown in an of kinase activity was present in Cdk2 and that been with cyclin A or cyclin E in the of Myt1, indicating that Cdk2·cyclin A Cdk2·cyclin E complexes were by when Cdc2 was for Cdk2 in the cyclin A complex, Myt1 reduced kinase as with or the the Cdk not the cyclin is a key for a particular Cdk·cyclin complex is by the Myt1 kinase. To Myt1 substrate specificity of Myt1, Wee1, and Cdk·cyclin kinases were in an in kinase containing by analysis of shown in Myt1 and Wee1 were capable of phosphorylating Cdc2 as well as a Cdc2 of which was with cyclin The was by Myt1 or Wee1, indicating that these kinases were phosphorylating Cdc2 on residue Thr14 Direct analysis and analysis using that human Myt1, Tyr15 phosphorylated Cdc2 on Thr14 not with of human and Xenopus Myt1 (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google Scholar). Cdk2·cyclin complexes were used as substrates in these kinase we found that Myt1 to Cdk2 that was with A or as well as Cdk4 with cyclin In Wee1 phosphorylated Cdk2 with A or but not Cdk4 with cyclin as has been N. Broome M. Hunter T. EMBO J. 1995; 14: 1878-1891Crossref PubMed Scopus (373) Google Scholar). using a or an in kinase with we observed that Myt1 only phosphorylated and Cdk·cyclin complexes in which Cdc2 was the catalytic subunit. To Myt1 phosphorylation of Cdc2 cyclin we a Myt1 kinase using Cdc2 as shown in Myt1 did not of the Cdc2·cyclin B1 complex, by Cdc2 with cyclin Myt1 to indicating that Myt1 only Cdc2 that is with a cyclin subunit. The Wee1 kinase was capable but this phosphorylation was when the was which is with L.L. P.J. M.J. F. Piwnica-Worms H. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). To the regulation of endogenous Myt1, were corresponding to Myt1 residues and These are and used for of cell and membrane fractions from human of the a membrane-associated This with baculovirus-expressed human Myt1 as well as Myt1 that was in an in not In Xenopus mitotic the of is concomitant with Myt1 hyperphosphorylation and decreased kinase activity (29Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (548) Google Scholar). To human Myt1 exhibits similar cell cycle we examined Myt1 in from human cells as well as cells that been and M phases of the cell cycle by with and analysis using and that Myt1 to a in mitotic The to this is phosphatase of the mitotic the of Myt1 to that observed in and extracts Myt1 were in the mitotic indicating that residues are phosphorylated during Further, analysis of and membrane fractions from cells that human Myt1 membrane-associated during M-phase to the Myt1 in mitotic but was capable of Myt1 in from as well as or cells of the mitotic this Myt1 that this only Myt1 not with the this was of mitotic Myt1 Myt1 likely the F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (273) Google it is that membrane is for Myt1 hyperphosphorylation observed during To investigate this we a cell that a non-membrane-associated Myt1 which the (see This cell was in mitosis with and the and fractions were analysis of these fractions that the a reduced similar to that for the endogenous Myt1 present in mitotic This that a in the cytosol that is capable of phosphorylating Myt1 during of Myt1 with We the that this kinase is also present in the To the kinase activity of endogenous human Myt1, from cell extracts were for to Cdk·cyclin complexes. A that the were capable of Myt1 from from and mitotic as the to mitotic with analysis of Myt1, reduced the kinase activity of Cdc2·cyclin B1 and B1 complexes, B1 complexes, and on the B1 complex to Cdk2·cyclin A and Cdk2·cyclin E complexes These results that the endogenous Myt1 kinase phosphorylates Cdc2 on residue Thr14 and not To the kinase activity of human Myt1 was cell we the kinase activity of Myt1 from and Myt1 Myt1 from mitotic extracts approximately reduced kinase activity Analysis of Myt1 using an Myt1 that Myt1 was in as well as and cells this to Myt1 from These results suggest that Myt1 is during and G2 but inactive during M concomitant with Myt1 The of cell kinase activity and phosphorylation for human and Xenopus Myt1 that Myt1 kinases may be regulated by a conserved an of this we the of human Myt1 that been in Xenopus and mitotic egg shown in 7 mitotic extracts an activity that reduced the of of the human Myt1 proteins The reduced observed for Myt1 the residues and that phosphorylated residues responsible for the Myt1 are not within of these the Xenopus mitotic extracts were from egg extracts that been an M-phase by of cyclin it is that Cdc2·cyclin B1 may Myt1 as an as has been proposed for and Wee1 P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (270) Google Scholar, T. J.L. Mol. Biol. Cell. 1995; 6: PubMed Scopus Google Scholar). To this we first determined Cdc2·cyclin B1 affect the of a Myt1 Sf9 containing Cdc2·cyclin B1 were with a in the of an ATP analysis of this that the with a reduced after Cdc2·cyclin B1 7 to a observed for Myt1 in mitotic extracts from human cells not 7 B also that the Myt1 a reduced to Myt1 that been phosphorylated by the Cdc2·cyclin B1 kinase. The phosphorylation of Myt1 by Cdc2·cyclin B1 was by including in an in kinase that Myt1 and Cdc2·cyclin B1 7 analysis that Cdc2·cyclin B1 phosphorylated Myt1 on threonine and residues not an Sf9 we examined the activity of Myt1 was by Cdc2·cyclin B1 phosphorylation. A was first with a B1 by of Cdc2·cyclin The Cdc2·cyclin B1 complex was by and its kinase activity was determined by a kinase We observed that phosphorylation of Myt1 by B1 affect on its to Cdc2 kinase activity not phosphorylation by Cdc2 not to Myt1 catalytic activity. In this we several biochemical properties of the human Myt1 kinase. We found that human Myt1 was capable of phosphorylating and Cdc2 with cyclin A or cyclin In Myt1 was to or Cdk·cyclin complexes that function in the cell In Myt1 did not Cdk4·cyclin Cdk2·cyclin or Cdk2·cyclin A complexes. The of Myt1 to Cdk4 was not Cdk4 an residue the corresponding Thr14 Myt1 phosphorylated Cdc2·cyclin A complexes, Myt1 specificity to be determined principally by the Cdk subunit. In Cdk2 Tyr15 is phosphorylated to Thr14 (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar, 12Sebastian B. Kakizuka A. Hunter T. Proc. Natl. Acad. Sci. U. S. A.. 1993; 90: 3521-3524Crossref PubMed Scopus (215) Google with in results that Myt1 not Cdk2 on It is that Myt1 Cdk2 that has been phosphorylated on Tyr15, as has been (11Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (724) Google Scholar). kinases may be responsible for phosphorylating Thr14 in as a non-membrane-associated Thr14 kinase activity that is present in bovine thymus cytosol (31Matsuura I. Wang J.H. J. Biol. Chem. 1996; 271: 5443-5450Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In the of Tyr15 phosphorylation on endogenous Cdk2 that Wee1, or another kinase, is the major kinase activity that Cdk2·cyclin kinase activity during the G1/S The kinase activity of human Myt1 was decreased during M-phase This decreased activity with and of phosphorylation of the of and in human Myt1, 10 and for the Myt1 hyperphosphorylation observed during M of these to the Cdc2·cyclin B1 phosphorylation Z. S. N. Piwnica-Worms H. Biol. 1994; 4: Full Text Full Text PDF PubMed Scopus Google with that Cdc2·cyclin B1 phosphorylated Myt1 in that an but not M-phase Myt1, or Myt1 phosphorylated by Cdc2·cyclin B1 in vitro, that a residue the is phosphorylated during phosphorylates Myt1 during in that Cdc2·cyclin B1 Myt1 on that its on While this phosphorylation did not affect Myt1 kinase we the that, in Cdc2 phosphorylation of Myt1 may be a for that Myt1 catalytic activity. the in Myt1 gel by Cdc2·cyclin B1 phosphorylation that an may be to the of Myt1 regulation during the cell We and for in the human Myt1 We are also to the for Cdk2·cyclin A and Cdk2·cyclin E Morgan of for Cdc2 and Cdk2 clones and for cyclin for Xenopus extracts and and for and and for and the proteins used throughout this