Rescue of a Human Cell Line from Endogenous Cdk1 Depletion by Cdk1 Lacking Inhibitory Phosphorylation Sites

Abstract

Cells that transiently overexpress cyclin-dependent kinase 1 lacking inhibitory phosphorylation sites (Cdk1-AF) undergo premature and catastrophic mitosis, reflecting the key role for Cdk1 in promoting a timely transit from G2 into mitosis. Conversely, cells depleted of Cdk1 undergo repeated S phases without intervening mitoses (endoreduplication), reflecting a role for Cdk1 in preventing premature S phases. It is not known how Cdk1 prevents entry into S phase at times in G2 when it does not promote mitosis. Also uncertain is the extent of redundancy between inhibitory phosphorylation and other mechanisms for controlling Cdk1 activity. We describe here human cells that not only tolerate stable Cdk1-AF expression but also rely on it for survival when endogenous Cdk1 is depleted. When residual endogenous Cdk1 expression is further depleted, however, proliferation of Cdk1-AF-rescued cells is inhibited. Interestingly, this inhibition is not accompanied by endoreduplication. These results are consistent with a two-threshold model for Cdk1 kinase activity, one for suppressing endoreduplication and one for promoting mitosis. They also indicate that inhibitory phosphorylation is indispensable for only a fraction of the total cellular complement of Cdk1. Cells that transiently overexpress cyclin-dependent kinase 1 lacking inhibitory phosphorylation sites (Cdk1-AF) undergo premature and catastrophic mitosis, reflecting the key role for Cdk1 in promoting a timely transit from G2 into mitosis. Conversely, cells depleted of Cdk1 undergo repeated S phases without intervening mitoses (endoreduplication), reflecting a role for Cdk1 in preventing premature S phases. It is not known how Cdk1 prevents entry into S phase at times in G2 when it does not promote mitosis. Also uncertain is the extent of redundancy between inhibitory phosphorylation and other mechanisms for controlling Cdk1 activity. We describe here human cells that not only tolerate stable Cdk1-AF expression but also rely on it for survival when endogenous Cdk1 is depleted. When residual endogenous Cdk1 expression is further depleted, however, proliferation of Cdk1-AF-rescued cells is inhibited. Interestingly, this inhibition is not accompanied by endoreduplication. These results are consistent with a two-threshold model for Cdk1 kinase activity, one for suppressing endoreduplication and one for promoting mitosis. They also indicate that inhibitory phosphorylation is indispensable for only a fraction of the total cellular complement of Cdk1. Cyclin-dependent kinases (Cdks) 4The abbreviations used are: Cdk, cyclin-dependent kinase; IPTG, isopropyl 1-thio-β-d-galactopyranoside; siRNA, small interfering RNA; MPA, mycophenolic acid; MPA/X, mycophenolic acid and xanthine; HA, hemagglutinin; WT, wild type. control eukaryotic cell cycle progression, ensuring that each phase occurs in an orderly and timely manner (1.Nigg E.A. Nat. Rev. Mol. Cell Biol. 2001; 2: 21-32Crossref PubMed Scopus (1252) Google Scholar, 2.Nurse P. Nature. 1990; 344: 503-508Crossref PubMed Scopus (2225) Google Scholar, 3.Nurse P. Chembiochem. 2002; 3: 596-603Crossref PubMed Scopus (105) Google Scholar). To be active, Cdks must be bound to an appropriate cyclin molecule and free from inhibition by Cdk inhibitors. Their action can be further controlled by changes in subcellular localization and phosphorylation state. Cdks therefore have a variety of ways in which their activity can be modulated in response to extrinsic (e.g. presence of growth factor) or intrinsic (e.g. completion of DNA synthesis) cues (4.Malumbres M. Barbacid M. Trends Biochem. Sci. 2005; 30: 630-641Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar, 5.Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1803) Google Scholar, 6.Obaya A.J. Sedivy J.M. Cell Mol. Life Sci. 2002; 59: 126-142Crossref PubMed Scopus (338) Google Scholar, 7.Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar, 8.Takizawa C.G. Morgan D.O. Curr. Opin. Cell Biol. 2000; 12: 658-665Crossref PubMed Scopus (313) Google Scholar). Transitions from G2 to mitosis and from G1 into S phase are key cell cycle control points. Simple eukaryotes have a single Cdk (Cdk1, or Cdc2 and Cdc28 in budding and fission yeast, respectively) that controls both of these transitions. Higher eukaryotes have multiple Cdks (Cdk1 to Cdk11) with more specialized roles, but Cdk1 and its role in promoting mitosis have been conserved. In the normal cell cycle, mitotic cyclins (cyclins A, B, and B3) accumulate during G2, and Cdk1 is held in a relatively inactive state by inhibitory phosphorylation at residues Tyr15 (in fission yeast) or Thr14 and Tyr15 (higher eukaryotes). Two kinases (Myt1 and Wee1) and one phosphatase (Cdc25) determine the phosphorylation status of these residues, and, together with Cdk1, these are thought to constitute a molecular switch that commits cells to undergo mitosis. Thus, in late G2, the sudden increase in Cdk1 activity that coincides with entry into mitosis is thought to be achieved by a double positive feedback loop in which Cdk1 kinase activity both inhibits Wee1 kinase and activates Cdc25 phosphatase (9.O’Farrell P.H. Trends Cell Biol. 2001; 11: 512-519Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The resulting flood of Cdk1 activity promotes mitosis by phosphorylating a range of nuclear substrates (e.g. lamins and histone H3) until its mitotic partner, cyclin B, is degraded at anaphase. The intrinsic cellular signals that determine when this switch is turned on during a normal cell cycle are unclear. The in vivo importance of inhibitory phosphorylation of Cdk1 has been demonstrated by experiments involving mutant Cdk1 proteins lacking phosphorylation sites. Fission yeasts with a Y15F mutation advance prematurely into mitosis (10.Gould K.L. Nurse P. Nature. 1989; 342: 39-45Crossref PubMed Scopus (854) Google Scholar), whereas vertebrate cells overexpressing Cdk1 with T14A and Y15F substitutions (Cdk1-AF) undergo mitotic catastrophe (i.e. entry into mitosis before DNA replication is complete, resulting in aberrant mitosis and cell death) (11.Krek W. Nigg E.A. EMBO J. 1991; 10: 3331-3341Crossref PubMed Scopus (249) Google Scholar, 12.Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar). In a variety of systems, mutation of Cdk1 inhibitory phosphorylation sites also impairs G2 arrest in response to genotoxic damage, indicative of signaling pathways that sense such damage and culminate in the inhibitory phosphorylation of Cdk1 (13.Blasina A. Paegle E.S. McGowan C.H. Mol. Biol. Cell. 1997; 8: 1013-1023Crossref PubMed Scopus (79) Google Scholar, 14.Fletcher L. Cheng Y. Muschel R.J. Cancer Res. 2002; 62: 241-250PubMed Google Scholar, 15.Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (251) Google Scholar). Despite the crucial role that inhibitory phosphorylation of Cdk1 undoubtedly plays in regulating the G2/M transition, it remains unclear whether the autocatalytic burst of Cdk1 activity that accompanies Cdk1 dephosphorylation is absolutely required to promote the G2/M transition or whether relief from other inhibitory mechanisms can suffice. Another conserved role for Cdk1 is to ensure that S phase is dependent on completion of a preceding mitosis. This role was first revealed in fission yeast after inactivation of Cdk1/cyclin B; cells failing to enter mitosis do not simply arrest in G2 but reset in G1 and enter successive but discrete rounds of S phase, leading to cells with ploidies of 4, 8, 16 N, etc. (16.Correa-Bordes J. Nurse P. Cell. 1995; 83: 1001-1009Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 17.Hayles J. Fisher D. Woollard A. Nurse P. Cell. 1994; 78: 813-822Abstract Full Text PDF PubMed Scopus (332) Google Scholar, 18.Moreno S. Nurse P. Nature. 1994; 367: 236-242Crossref PubMed Scopus (303) Google Scholar). A similar endoreduplication phenotype was observed when Cdk1 was depleted in human cells by genetic means (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). In fission yeast, Cdk1 binds at replication origins and prevents the formation of prereplication complexes, apparently by phosphorylating key components of the prereplication complex (20.Wuarin J. Buck V. Nurse P. Millar J.B. Cell. 2002; 111: 419-431Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and various components of the mammalian prereplication complex are also known to be phosphorylated by Cdk1 (21.Blow J.J. Dutta A. Nat. Rev. Mol. Cell Biol. 2005; 6: 476-486Crossref PubMed Scopus (532) Google Scholar, 22.Diffley J.F. Curr. Biol. 2004; 14: R778-R786Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Thus, assuming that the serine kinase activity of Cdk1 is required to suppress endoreduplication during S and G2, the question arises of how this is achieved without promoting premature mitosis. To address questions of this kind, it is useful to have a genetic system in which the functionality of variant Cdk1 proteins can be analyzed in cells depleted for endogenous Cdk1. The human HT2-19 cell line is uniquely suited to this purpose (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). HT2-19 cells have one inactivated CDK1 allele and one allele that is regulated by the lac repressor. In the presence of IPTG, Cdk1 protein levels and kinase activity in HT2-19 cells are 30–40% of those in parental HT1080 cells but sufficient for cell proliferation, albeit with an increased doubling time. After removal of IPTG, Cdk1 protein levels and kinase activity decline to <10% of +IPTG values, and appreciable endoreduplication is observed, accompanied by apoptosis. Because they can be rescued from IPTG dependence by transfection with a Cdk1 expression plasmid (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar), HT2-19 cells can be used in complementation tests to assess the functionality of variant Cdk1 proteins. In the present study, we find that the Cdk1 from Schizosaccharomyces pombe (Cdc2) cannot rescue HT2-19 cells from IPTG dependence, whereas human Cdk1-AF can. Further analysis reveals that the latter complementation requires a small amount of residual endogenous Cdk1 expression, without which cells fail to divide or endoreduplicate. These results indicate that some, but not all, cellular Cdk1 must be susceptible to inhibitory phosphorylation for a successful G2 to M transition. The results also suggest a model in which two thresholds of Cdk1 activity must be exceeded sequentially during the cell cycle, the first to inhibit endoreduplication and the second to promote entry into mitosis. Cell Culture and Electroporation−HT1080 and HT2-19 cell culture and stable transfection by electroporation using a Gene Pulser electroporator (Bio-Rad) were (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). was to at 1 To for with was to the after IPTG was to the to a of and have been (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). The of the CDK1 in was to to an and were was with and the Cdk1 was and the was to the of or by of the Cdk1 D. Gu Y. Morgan D.O. Mol. Biol. Cell. 3: PubMed Scopus Google Scholar). To a from by and the of were and To and a from was to or the and of CDK1 were by and DNA A CDK1 was by an from into (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar), at an at the of CDK1 kinase were (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar), using a by to the human Cdk1 or a of cell were (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). Cdk1, a and a were a and were analysis of for DNA was (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). Gene CDK1 was at a and into were by a for at the CDK1 allele using and was on of cells (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). To determine whether the CDK1 allele been endogenous Cdk1 expression, were analyzed by with Cdk1 for endogenous CDK1 and or and were from and to cells by to the cells were at in or in the before HT2-19 cells were in with HT2-19 were in without The a and in was to the and to cells or with of to a of and of cells was (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). Cdk1-AF HT2-19 Cells from Cdk1 plasmid the human Cdk1 by the CDK1 and the which to mycophenolic acid and was to rescue HT2-19 cells from IPTG dependence (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). of were to human Cdk1 with a and with or without T14A and Y15F A further was to the only Cdk of S. complementation experiments were to one or more of these or only the for their to rescue HT2-19 cells from IPTG HT2-19 cells were for expression (in and or for both expression and IPTG (in IPTG in HT2-19 (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google by after transfection with to but was to the of IPTG by Cdk1 Thus, levels of were by and, by When a of the of IPTG by and were similar and in experiments 1 and the of the complementation and therefore to for Cdk1 with similar The expression plasmid for Cdc2 was to levels of IPTG This was when of of the of in the of IPTG, of of the by were to the S. pombe protein tests in of HT2-19 cells of in the of the cells were used for each of in a of those in of in by of cells not of HT2-19 cells of in the of the cells were used for each of in a of those in of in by of cells not in a and in HT2-19 that Cdk1 was in and that endogenous Cdk1 expression was from were analyzed by with a Cdk1 from C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar, 15.Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (251) Google Scholar, Mol. Biol. Cell. 3: PubMed Scopus Google Scholar), both endogenous and Cdk1 were a of the latter more the of the and, for with expression lacking the two that and Thus, were in whereas only the of these were in In endogenous Cdk1 were also from Cdk1 by their dependence on A (e.g. and however, expression of endogenous Cdk1, of a (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). Interestingly, of the (e.g. and more endogenous Cdk1, this was not the for of the the amount of protein to endogenous Cdk1 was similar between consistent with the that expression of is not by whereas a is required to for endogenous Cdk1 These results the from the complementation analysis that is of for the Cdk1 in HT2-19 cells in the of in HT2-19 that Cdk1 was in the rescued histone kinase were on of two and and two and that been with or without an with the mitotic of not of kinase activity were in but the activity was increased by of and whereas such increase was for and results were when were and with a These results that endogenous Cdk1, is by of inhibitory phosphorylation entry into mitosis, whereas is kinase activity, in was similar in rescued but and in from cells The Cdk1 does not Cdk1, it a that is by the of the The that the activity before is similar in rescued is consistent with the similar of to endogenous Cdk1 in these by HT1080 Cells but of to complement the Cdk1 in HT2-19 cells was a the the of Cdk1-AF To determine whether the of expression was a of the parental HT1080 cells the A.J. Porter A.C.G. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google were with and an expression for and expression for or After transfection of of expression by removal of increased the of This can be of the to the expression also expression of the After transfection of however, of expression the of of and in the of (i.e. or were the to levels of whereas the latter of be in of and in the presence of (i.e. to suppress expression from or and without for during which cells normal and to these suggest that HT1080 cells levels of are to but that they are at a and by in which expression is The expression of in HT2-19 in the of IPTG is therefore these a to with or without CDK1 of cells of cells in a of HT2-19 rescued and were for further but and more when IPTG was to the 4, A and This that for such is a in the of IPTG to cells that their endogenous Cdk1 expression, we HT2-19 cells were of at (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). This endogenous Cdk1 expression in IPTG dependence at the of first analysis (e.g. and It also we that such endogenous Cdk1 was when first this dependence in The that expression of is in such that its expression in HT1080 cells is that Cdk1 activity remains to HT2-19 cells when endogenous Cdk1 is This is consistent with the in the presence of IPTG, the cell doubling and amount of Cdk1 protein are both in HT2-19 with parental HT1080 cells (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar). on on the it was that residual endogenous Cdk1 expression in the of IPTG, when to HT2-19 with Cdk1 to rescue HT2-19 We therefore to whether residual endogenous Cdk1 expression was required for not to rescue HT2-19 cells from IPTG To we to the CDK1 allele in by of a to that used to the other allele (19.Itzhaki J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google but with a were by for but of the the allele We therefore used a interfering to expression of endogenous CDK1 without expression of the Cdk1 of and cells with or a control that endogenous but not Cdk1 was depleted by the for of a The of on cell proliferation, and were and to be for each cell line HT2-19 cells in IPTG and with cell proliferation, and increased with cells with control HT2-19 cells therefore to they do to removal of In the proliferation, and of cells were relatively by whereas in cells proliferation was but with of cell or These results suggest in the of residual endogenous Cdk1, is to promote mitosis but to suppress whereas is of both was also after transfection by of and of cells and similar to those that after IPTG from were in but not HT2-19 the however, similar of endoreduplication was not in of the HT2-19 and it was that cell proliferation was by in and but not in or The here that Cdk1 that inhibitory phosphorylation sites is to rescue a human cell line by of its endogenous Cdk1. its this is a for at two the sudden from inhibitory phosphorylation is a and conserved of the G2/M transition for the normal G2/M transition. experiments have that of the inhibitory phosphorylation of Cdk1 to mitotic catastrophe and not therefore be with cell (11.Krek W. Nigg E.A. EMBO J. 1991; 10: 3331-3341Crossref PubMed Scopus (249) Google Scholar, 12.Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar). however, other mechanisms for Cdk1 activity, such Cdk and and these in be used to premature mitosis by Cdk1 that is not to inhibitory the levels of Cdk1-AF expression used to mitotic catastrophe were for the that more levels can be levels of Cdk1-AF were relatively in at in the (13.Blasina A. Paegle E.S. McGowan C.H. Mol. Biol. Cell. 1997; 8: 1013-1023Crossref PubMed Scopus (79) Google Scholar, 15.Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (251) Google Scholar). the of the the be that inhibitory phosphorylation a during the normal mammalian cell cycle, has been for budding yeast A. Nature. PubMed Scopus Google Scholar). We not an role for inhibitory however, residual endogenous Cdk1 kinase activity that was to inhibitory phosphorylation was in Cdk1-AF-rescued cells We the that such residual activity was in with the Cdk1-AF kinase activity, for survival of such To we to the Cdk1 allele in but only that at the other Cdk1 during of HT2-19 this until we be that were we used to residual endogenous Cdk1 in a Cdk1-AF-rescued and and, in and proliferation of and but not and and that residual Cdk1 activity is in Cdk1-AF-rescued This can be the increase in activity by dephosphorylation of the residual endogenous Cdk1 in such is sufficient to a G2 of activity required for entry into mitosis only when with the activity by Cdk1-AF Thus, from inhibitory phosphorylation of Cdk1 is required for the G2 to M transition but only to a fraction of the total cellular Cdk1, a of Cdk1 activity is It is to of endogenous Cdk1 activity in HT2-19 the endoreduplication was by a similar of Cdk1-AF-rescued cells and This can be in of a second G2 of Cdk action which endoreduplication is In this of the rescued is not to G2 Cdk1 activity This two-threshold model is to the model by and Nurse Nurse P. Trends Genet. 1996; 12: Full Text PDF PubMed Scopus Google to how a single Cdk1/cyclin in yeast promote both S phase and M phase in In that Cdk1 activity, cyclin in late mitosis, promotes S and M phases when and activity thresholds are in the role of Cdk1 in promoting S phase has been by other Cdk whereas its role in preventing endoreduplication to have been conserved. The by which Cdk1 prevents endoreduplication is uncertain but is from the that prevents multiple replication a single S phase, cells S phases J. Fisher D. Woollard A. Nurse P. Cell. 1994; 78: 813-822Abstract Full Text PDF PubMed Scopus (332) Google Scholar, J.E. Gilbert C.S. Porter A.C.G. Nat. Genet. 1997; 15: 258-265Crossref PubMed Scopus (138) Google Scholar, A. S. A. Y. Porter A.C.G. M. Mol. Biol. Cell. 14: PubMed Scopus Google Scholar). In fission yeast, the requires Cdk1 to the of replication it the of replication origins by phosphorylating of replication complex such (20.Wuarin J. Buck V. Nurse P. Millar J.B. Cell. 2002; 111: 419-431Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). assuming that a similar in mammalian cells and that a of Cdk1 activity is we cannot only the results we have observed but also a to the question of how Cdk1 kinase activity can suppress endoreduplication without promoting mitosis. it is consistent with of the this two-threshold model remains and further the of this is required to its in more A L. M. L. Cell PubMed Scopus Google the formation of of Cdk1 by in that be redundancy between Cdk1 and It is that in HT2-19 cells in response to the of one CDK1 allele or to of the such activity however, to for the of Cdk1 in HT2-19 cells when IPTG is is to a role in rescued by expression of or the Cdk1 with at the of Cdk1 was by its to complement a mutation in fission yeast Nurse P. Nature. PubMed Scopus Google Scholar), a of between the yeast and human proteins. we were to the and that was to complement HT2-19 cells without Further is required to determine whether this an of the yeast protein to with (e.g. cyclin in human cells or whether it can that are for premature entry into S The latter is not only by the that the yeast protein in the G1 to S transition in its normal but also in the of mammalian Cdk1 can with cyclin and promote the G1 to S transition P. Nat. Cell Biol. 2005; PubMed Scopus Google Scholar). The of Cdk1 inhibitory phosphorylation here were of a more analysis of the of HT2-19 cells for the in vivo analysis of variant Cdk1 in vivo of Cdk have expression in yeast Cdk W. J. Nigg E.A. V. J. Cell Sci. PubMed Scopus Google Scholar, J. P. Nurse P. J. Cell Sci. 1994; PubMed Google or in mammalian cells that to endogenous Cdk1, both of which have (11.Krek W. Nigg E.A. EMBO J. 1991; 10: 3331-3341Crossref PubMed Scopus (249) Google Scholar, 12.Norbury C. Blow J. Nurse P. EMBO J. 1991; 10: 3321-3329Crossref PubMed Scopus (399) Google Scholar, 15.Jin P. Gu Y. Morgan D.O. J. Cell Biol. 1996; 134: 963-970Crossref PubMed Scopus (251) Google Scholar, P. S. Morgan D.O. J. Cell Biol. PubMed Scopus Google Scholar). HT2-19 cells variant Cdk1 to be in cells depleted of endogenous Cdk1. Thus, we were to for the first whether yeast Cdk1 (Cdc2) or human Cdk1-AF for Cdk1. The results the HT2-19 complementation system to be and In the of Cdk1-AF however, the results were by the of residual Cdk1 activity in the of these to be in of mutant Cdk1 from a cell line in which Cdk1 can be more depleted. This can be achieved by the of a more system the used in HT2-19 such S. M. W. Sci. S. A. 2000; PubMed Scopus Google Scholar), used for the analysis of mutant in HT1080 cells A.J. Porter A.C.G. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar). We are to for of the with with