Journal of Cell Science 112, 3361-3371 (1999) 3361 Printed in Great Britain © The Company of Biologists Limited 1999 JCS0474

The C-terminal domain of the Cdc2 inhibitory kinase Myt1 interacts with Cdc2 complexes and is required for inhibition of G2/M progression

Nicholas J. Wells1,*,‡, Nobumoto Watanabe2, Tsuyoshi Tokusumi2, Wei Jiang1, Mark A. Verdecia1 and Tony Hunter1,‡ 1Molecular Biology and Virology Laboratory, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037, USA 2Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305- 0074, Japan *Present address: Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK ‡Authors for correspondence

Accepted 16 July; published on WWW 22 September 1999

SUMMARY

Activation of Cdc2, is the universal event controlling the dependent manner. Truncation of the C-terminal domain onset of mitosis. In higher eukaryotes, Cdc2 activity is in of Myt1 prevented its ability to induce G2/M phase arrest part regulated by inhibitory phosphorylation of Thr14 and in overexpression studies in human cells and dramatically Tyr15, catalyzed by Wee1 and Myt1, which prevents reduced its ability to phosphorylate Cdc2 in vitro. We catastrophic premature entry into mitosis. In this study we demonstrate that the C-terminal domain of Myt1 was defined the function of Myt1 by overexpression studies in required for recruitment of Cdc2, and we infer that this both S. pombe and a human osteosarcoma cell line. Similar domain lies in the cytoplasm because it can interact with to Wee1, overexpression of human Myt1 prevented entry and is phosphorylated by Cdc2. In conclusion, we propose into mitosis in both cell types; however, Myt1 catalytic that Myt1 can negatively regulate Cdc2/cyclin B1 and activity was not essential for the cell cycle delay observed inhibit G2/M progression by two means, both of which with human cells. Myt1 expression was restricted to require the C-terminal domain; first, Myt1 can bind and proliferating cells. Furthermore, we detected no major sequester Cdc2/cyclin B1 in the cytoplasm preventing entry decline in Myt1 abundance prior to the entry into into the nucleus, and, second, it can phosphorylate mitosis, which coincides with the loss of Myt1 activity. We associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus localized mitotic phosphoepitopes, recognized by the inhibiting its catalytic activity. monoclonal antibody MPM-2, to the C-terminal domain of Myt1. The mitotic peptidyl-prolyl isomerase, Pin1, was able to associate with this domain in a phosphorylation- Key words: Myt1, Cdc2, Mitosis

INTRODUCTION et al., 1991). Dephosphorylation of Thr14 and Tyr15 upon activation of the dual-specificity phosphatase, Cdc25, coupled In all eukaryotic cells, progression through the cell cycle is with inactivation of the Thr14 and Tyr15 kinases (McGowan regulated by the sequential activation of the cyclin-dependent and Russell, 1995; Parker et al., 1995; Watanabe et al., 1995; protein kinases (CDKs) (Norbury and Nurse, 1992; Morgan, Mueller et al., 1995; Liu et al., 1997; Booher et al., 1997) 1997). Activation of the p34cdc2 (Cdc2) CDK is the universal results in the precipitate activation of cyclin B/Cdc2 which event controlling the onset of mitosis. The ability of Cdc2 to triggers M phase. In S. pombe, phosphorylation of Tyr15 is induce M phase is dependent on its association with a cyclin catalyzed by the Wee1 and Mik1 kinases; the additional level partner, yet is also further regulated in a positive and negative of inhibitory phosphorylation, at least under normal conditions, fashion by phosphorylation. Specifically, phosphorylation at at Thr14 exists only in higher eukaryotes. Phosphorylation of Thr161 by the CDK activating kinase (CAK) is necessary Thr14/Tyr15 has been implicated in the G2/M checkpoint that for activation of the CDK/cyclin complex, whereas blocks entry into mitosis in the presence of damaged DNA (Jin phosphorylation of Thr14 and Tyr15 by Wee1 and Myt1 et al., 1996; Poon et al., 1997) or incomplete DNA replication, kinases maintains the complex in an inactive state. Indeed, in although a cyclin B/Cdc2 inhibitory factor may also be higher eukaryotes there is a gradual cytoplasmic accumulation important (Kumagai and Dunphy, 1995). of potentially active cyclin B/Cdc2 heterodimers during S and A dual-specificity, membrane-associated protein kinase G2, that are phosphorylated at Thr161 but maintained in the activity, able to phosphorylate Cdc2 on both Thr14 and inactive state by phosphorylation at Thr14 and Tyr15 (Norbury Tyr15 was identified in Xenopus and HeLa cell extracts 3362 N. J. Wells and others

(Kornbluth et al., 1994; Atherton et al., 1994) and the associate with its substrate, Cdc2/cyclin B1, which is Thr14/Tyr15 kinase, Myt1, was subsequently cloned necessary for Myt1 to phosphorylate Cdc2, and also anchors (Mueller et al., 1995; Liu et al., 1997). In contrast to Wee1, Cdc2 within the cytoplasmic compartment. Because this which is localized in the nucleus (McGowan and Russell, domain is phosphorylated by and interacts with Cdc2/cyclin 1995), Myt1 is localized to the endoplasmic reticulum (ER) B1, we deduce that it must lie in the cytoplasm. In and Golgi complex by a membrane-targeting domain on the conclusion, we provide evidence that the C-terminal domain C-terminal side of the catalytic domain (Liu et al., 1997). is required in combination with the catalytic domain for the Specific subcellular localization of protein kinases is of biological activity of Myt1 in inhibiting G2/M progression. significant importance; indeed the compartmentalization modulated by a nuclear export sequence of cyclin B1 regulates the physiological activity of Cdc2/cyclin B1 (Li et MATERIALS AND METHODS al., 1997; Hagting et al., 1998; Yang et al., 1998). Myt1 exhibits a more restricted substrate specificity than Wee1, in Antibodies that it phosphorylates Cdc2/cyclin complexes but not α α Cdk2/cyclin complexes (Booher et al., 1997). This The 9E-10 - and 12CA5 HA monoclonal antibodies were utilized for immunoprecipitations and immunoblotting of Myc- and observation strongly suggests that Myt1 specifically HA-tagged respectively. Rabbit polyclonal antibodies to regulates G2/M phase transition through the inhibitory human Pin1 were used in the GST pull-down assay, as described (Lu phosphorylation of Cdc2. In turn, Myt1 itself is et al., 1996). Immunoblotting of human Cdc2 was carried out with hyperphosphorylated during mitosis, which is coincident rabbit polyclonal serum (antiserum 5517) as described (Watanabe et with its inactivation (Mueller et al., 1995; Booher et al., al., 1995). Antibodies C1-1 and R5084 against Myt1 were kindly 1997). Interestingly studies on Xenopus Myt1 have identified provided by Drs Booher and Piwnica-Worms, respectively (Booher et the kinase as an MPM-2 epitope containing protein (Mueller al., 1997; Liu et al., 1997). The monoclonal antibody MPM-2, was et al., 1995). The MPM-2 monoclonal antibody epitope is purchased from Upstate Biotechnology. a mitotic phosphorylation dependent motif, minimally Expression constructs, site-directed mutagenesis and phospho-Ser/Thr-Pro (Westendorf et al., 1994), and is DNA sequencing recognized by a monoclonal antibody raised by The human Myt1 expression vector, pcDNAmycMyt1, was a kind gift immunization with mitotic HeLa cell extracts (Davis et al., from Dr Piwnica-Worms (Liu et al., 1997). The Myt1 truncation 1983). series, in the mammalian expression vector pCS, was derived by PCR Recently a novel mitotic regulator, Pin1, a highly conserved amplification of the human Myt1 cDNA and cloned into the XbaI site. peptidyl-prolyl isomerase (PPIase) has been described, that The specific primers used were: Myt1 (full length) 5′ primer also recognizes the MPM-2 epitope (Ranganathan et al., 1997; AGAGAGTCTAGAGATGCTAGAACGGCCTCCTGC and 3′ primer Yaffe et al., 1997). Pin1 inhibits entry into mitosis when AGAGAGTCTAGATCAGGTTGGGTCTAGGGTGTC, ∆N-Myt1 5′ overexpressed in HeLa cells and microinjected into Xenopus primer AGAGAGTCTAGAGTCCTTCTTCCAGCAGA and 3′ primer two-cell stage embryos, yet is also required for progression as for full length Myt1, ∆C-Myt1 5′ primer as for full length Myt1 and 3′ primer AGAGAGTCTAGATCAGCCCAGGGGCTGTAGCC- through mitosis (Lu et al., 1996; Crenshaw et al., 1998; Shen ∆ ′ ∆ ′ et al., 1998). PPIases catalyze rotation around the peptide AGCTG and NC-Myt1 5 primer as for N-Myt1 and 3 primer as for ∆C-Myt1. pEGFPF utilized in cotransfections of U2-OS cells was bond preceding a proline residue and may regulate protein previously described (Jiang and Hunter, 1998). For the GST pull- folding and intracellular trafficking (for review see Schmid, down assay the Myt1 C-terminal domain was cloned to the pGEX- 1995). Phosphorylation of a Ser/Thr-Pro motif catalyzed by KG vector, Myt1 COOH-GST, at the BamHI and XhoI sites by PCR Cdc2/cyclin B could create a Pin1 substrate, enabling amplification using the 5′ primer AGAGAGGGATCCGCCAGCT- conformational changes driven by prolyl isomerization. The GGCTACAGCCCCTG and 3′ primer AGAGAGCTCGAGTCA- conformational change may alter enzymatic activity, or GGTTGGGTCTAGGGTGTC. Pin1 was cloned by PCR amplification modify the sensitivity to dephosphorylation by phosphatases into pEVRF0 (Janknecht, 1996), a mammalian expression vector that creating a timed phosphorylation induced activity/ contains an N-terminal HA tag, at the XmaI and Xba1 sites with the 5′ primer AGAGAGCCCGGGATGGCGGACGAGGAGAAGCTG modification. Thus Pin1, as well as Myt1, may play an ′ important role in the activation of Cdc2, pivotal for the and 3 primer AGAGAGTCTAGATCACTCAGTGCGGAGGATG- ATG. Mutagenesis of CDK consensus phosphorylation sites with the initiation of mitosis. C-terminal domain of Myt1 (described in the text) was carried out In this paper we examined the function and regulation of using the QuikChange site directed mutagenesis method (Stratagene) the biological activity of Myt1 and its interaction with the according to the manufacturer’s protocol. Constructs and mutations peptidyl-prolyl isomerase, Pin1. We demonstrate that were analyzed by automated DNA sequencing using an ABI Prism overexpression of Myt1 inhibits G2/M progression in the 377XL. eukaryotic cell cycle. Myt1 expression was restricted to proliferating cells, and furthermore there was no apparent Expression studies in S. pombe decrease in abundance following entry into mitosis, The S. pombe strain 1058 (h+, leu1-32, ura4-D18, ade6-210, his7- coincident with loss of Myt1 activity. We also localized the 366) was transformed by electroporation with pSLF273-Myt1, cell cycle specific MPM-2 epitopes within Myt1 to the C- pSLF273-Myt1(D/A) and pSLF273 alone (Forsburg and Sherman, 1997), containing the mid strength nmt41 promoter, under repressed terminal domain and show that Pin1 interacts with this conditions (5 µg/ml thiamine). Transformed cells were washed five domain in a phosphorylation-dependent manner. We times with thiamine free medium, inoculated into medium with and demonstrated that the G2/M arrest induced upon without thiamine and incubated for a further 24 hours. Cells were then overexpression of Myt1 requires its C-terminal domain. We harvested for examination by phase contrast microscopy and flow ascribe this to the ability of the C-terminal domain to cytometric analysis. Regulation of Myt1 3363

Transient transfections binding buffer (20 mM Tris-HCl (pH 8.0), 10% glycerol, 75 mM Human embryonal kidney 293T or human osteogenic sarcoma U2-OS NaCl, 2 mM DTT and 0.025% Tween-20), and incubated with cells were grown to 25% confluence and then transfected by the recombinant Pin1 (Ranganathan et al., 1997). The bound proteins calcium phosphate coprecipitation method. Cells were harvested 36- were washed three times and analyzed by SDS-PAGE, following 48 hours later for flow cytometric analysis, immunoblotting or addition of SDS-sample buffer. immunoprecipitation. Immunoprecipitation and in vitro Myt1 kinase assay Cell synchronization and cell cycle analysis Transfected 293T cells were lysed in 50 mM Hepes (pH 7.5), 150 mM HSF8 cells were synchronized by serum starvation. Briefly, NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA proliferating HSF8s were cultured in Dulbecco’s modified Eagle’s 1 mM DTT and also containing the phosphatase and protease medium (DMEM) supplemented with 10% fetal bovine serum, inhibitors described above, and immunoprecipitations carried out with glutamine and antibiotics in a humidified atmosphere containing 10% the 9E-10 antibody. The immunoprecipitations were washed in 0.05% CO2 at 37¡C. Cells were washed three times in phosphate buffered Tween-20, 25 mM Tris-HCl (pH 7.5), 150 mM NaCl before saline (PBS), and incubated for at least 30 hours in serum free resuspension in the kinase buffer 50 mM Tris-HCl (pH 7.5), 10 mM supplemented DMEM. Cells were released from the G0 block by MgCl2 and 1 mM DTT. The kinase reaction was carried out as above readdition of DMEM containing 10% fetal bovine serum. Upon with recombinant baculovirus purified catalytically inactive Cdc2 release samples were taken at the noted times. Ninety percent of the (K/R)/cyclin B, resolved by SDS-PAGE and analyzed by cells were harvested for lysis and subsequent immunoblotting. The autoradiography. remaining cells from each time point were subjected to flow cytometric analysis as follows: cells were fixed in 70% ethanol, rehydrated and then treated with RNase A (100 µg/ml final RESULTS concentration) and propidium iodide (40 µg/ml final concentration) in PBS for 30 minutes at 37¡C. Cell-cycle distribution of 104 cells Myt1 overexpression in U2-OS cells perturbs normal was then determined on a Becton Dickinson FACScan. For cell cell cycle progression synchronization studies with HeLa and U2-OS cells, cultures were Our initial aim was to investigate whether Myt1 either released from a G1/S block, obtained by a double thymidine arrest as described (Heintz et al., 1983) or released from an M phase overexpression in a human osteosarcoma cell line, U2-OS, block, obtained from a thymidine/nocodazole block as described would affect cell cycle progression. To assess the effect on (Kanemitsu et al., 1998). the cell cycle distribution, cells were transiently cotransfected with expression vectors for Myc-tagged Myt1 and a Immunoblotting and coimmunoprecipitation membrane-targeted form of GFP, EGFPF (Jiang and Hunter, Cells were lysed in either Nonidet P-40 lysis buffer (50 mM Tris-HCl 1998), as a marker of transfection. Cells were harvested 48 (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol), or RIPA hours later, and the DNA content of cells both negative and buffer. Buffers were supplemented with 1 mM phenylmethylsulfonyl µ positive for GFP, within the same population, was then fluoride, 0.2 mM DTT, 10 units/ml aprotinin, 20 g/ml leupeptin, 0.1 assessed by flow cytometric analysis. Expression of Myt1 mM p-nitrophenylphosphate, 10 nM microcystin, 1 mM sodium fluoride and sodium orthovanadate and 0.1 mM sn-glycerol 2- caused a significant increase in the G2/M population relative phosphate. Cell lysates were clarified by centrifugation and protein to either untransfected cells or cells transfected with GFP concentrations were determined using the Bio-Rad protein assay, as alone (Fig. 1a). The proportion of cells arrested was detailed in the manufacturer’s protocol. Immunoprecipitations were dependent on the level of Myt1 expression (data not shown, either performed with 12CA5 (αHA) or 9E-10 (αMyc) and Protein A and Table 1). However, it was unclear whether Myt1 beads (Repligen). The immune complexes or proteins were separated overexpression caused an increase in the G2/M phase by discontinous SDS/polyacrylamide electrophoresis, transferred to population by a block in cell cycle progression, or as a Immobilon-P membrane (Millipore) and then blotted with primary and consequence of acceleration of G1 and S phases. To address secondary antibodies and detected by enhanced chemiluminescence this we treated Myt1 transfectants with thymidine, to (Amersham) as detailed in the manufacturer’s protocol. synchronize cells at the beginning of S-phase, 12 hours prior Purification of Myt1 C-terminal domain GST fusion, to harvesting for flow cytometric analysis. If cells transfected phosphorylation and GST pull-down assay with Myt1 are already blocked in G2/M phase, the drug E. coli BL21 cells transformed with Myt1 GST-COOH were induced should have little or no effect. As shown in Fig. 1b, cells by IPTG (0.1 mM final concentration) for 4 hours at 18°C. Induced transfected with Myt1 and subsequently treated with cultures were harvested by centrifugation, resuspended in lysis buffer thymidine still had significant numbers of cells in G2/M (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 1 mM relative to cells transfected with GFP alone. Hence Myt1 β EGTA, 10% glycerol, 0.1% Nonidet P-40, 20 mM -mercaptoethanol, overexpression causes a G2/M phase arrest and not an µ 1 mM PMSF, 1 mM benzamidine, 10 units/ml aprotinin and 20 g/ml acceleration of G1 or S phases. leupeptin), lysed by sonication and clarified by centrifugation. GST Interestingly, transfection with catalytically inactive Myt1 fusion proteins were bound to glutathione-agarose by a batch mixing (Asp251Ala) (shown to lack activity by an in vitro kinase method, washed in lysis buffer and subsequently eluted from a column with 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 20 mM reduced assay, see Fig. 6c), also induced an accumulation of cells glutathione and 20 mM β-mercaptoethanol. Eluted proteins were then within the G2/M population, albeit less efficiently than wild- dialyzed against 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20% type Myt1 (Table 1), indicating that Myt1 kinase activity is not glycerol, 20 mM β-mercaptoethanol. GST fusion proteins were essential for the G2/M arrest phenotype at higher expression phosphorylated with recombinant baculovirus purified Cdc2/cyclin levels. B1 in kinase buffer (50 mM Hepes (pH 7.5), 20 mM MgCl2, 1 mM DTT, and either 10 µM ATP and 10 µCi [γ-32P]ATP or 100 µM ATP Overexpression of Myt1 perturbs normal S. pombe alone, at 30¡C for 30 minutes. Following phosphorylation GST fusion cell cycle progression proteins were bound to glutathione-agarose, washed three time in Next we examined whether Myt1 functions in a fashion 3364 N. J. Wells and others

a Negative Positive Table 1. Titration series of Myt1 and Myt1-D/A transfections %G1 46 %G1 52 %S 26 %S 25 Cell cycle distribution %G2 28 %G2 23 GFP Construct transfected G1 (%) G2 (%) G2/G1 Alone Counts Counts Vector alone 44 24 0.55 Myt1 (2 µg) 32 38 1.19 Myt1 (6 µg) 30 49 1.63 Myt1 (18 µg) 26 53 2.04 Myt1-D/A (2 µg) 41 32 0.78 DNA Content DNA Content Myt1-D/A (6 µg) 33 42 1.27 Myt1-D/A (18 µg) 33 44 1.33 %G1 47 %G1 35 %S 25 %S 18 Expression plasmids (20 µg total) containing titrated amounts (shown in %G2 28 %G2 47 parenthesis) of wild-type Myt1 or the catalytically inactive Myt1-D/A mutant GFP+ (Asp251Ala) were cotransfected into U2-OS cells with 1 µg of EGFPF and Counts Counts Myt1 empty vectors. Cells were subsequently harvested, stained with propidium iodide and the cell cycle of the transfected, GFP positive cells, was assessed by flow cytometric analysis. The percentage of cells in G1 and G2 phases, are shown. The ratio of G2/G1 is also included for comparison.

DNA Content DNA Content from the nmt promoter, was assessed. Expression of Myt1 generated very elongated cells with a single diffuse nucleus b Negative Positive (Fig. 2), indicative of an arrest at the G2/M transition. Flow %G1 76 %G1 69 cytometric analysis was also consistent with such a conclusion %S 18 %S 21 (data not shown). Expression of catalytically inactive Myt1 %G2 9 %G2 10 (Asp251Ala) failed to arrest cell proliferation (data not shown). GFP Hence, Myt1 kinase activity is required for the G2 arrest when Counts Counts Alone overexpressed in wild-type S. pombe. Continued growth of the cell following DNA replication, rather than entry into mitosis is also a hallmark of overexpression of the Wee1Hu catalytic domain in S. pombe (Igarashi et al., 1991). We conclude that DNA Content DNA Content +Thymidine Myt1, like Wee1, causes a G2 cell cycle arrest following overexpression in S. pombe. These studies suggest that %G1 72 %G1 44 %S 18 %S 23 negative regulation of Myt1 is necessary for normal cell cycle %G2 10 %G2 33 progression through G2/M. GFP+ Counts Counts Myt1 Myt1 is a proliferation marker To assess whether Myt1 is regulated at the protein level we examined the cell cycle phase-specific expression profile of Myt1. First, the cell cycle expression profile of Myt1 in the DNA Content DNA Content HSF strain (HSF8) of non-transformed, non-immortalized human diploid foreskin fibroblasts (W. Jiang, unpublished Fig. 1. Overexpression of Myt1 perturbs normal U2-OS cell cycle observations) was investigated. Cells were serum starved for progression. (a) Flow cytometric analyses of GFP-negative and -positive U2-OS cells cotransfected with 20 µg of a Myt1 expression 48 hours to induce synchronization/quiescence and released construct or control vector and the EGFPF construct (5 µg). The from the G0 block by readdition of fetal bovine serum. Cells propidium iodide signal was used as a measure of DNA content to were subsequently harvested at intervals for cell cycle analysis determine the cell cycle profiles (G1, S and G2/M). The DNA by flow cytometry (Fig. 3a) and western blotting analysis with histograms from each assay contain data from 104 cells. The αMyt1 and αCdc2 antisera. Quiescence, induced by either percentage of cells in each phase of the cell cycle is shown. (b) as (a) serum starvation (Fig. 3a, upper panel), or contact inhibition at except cells were treated with thymidine prior to harvesting. confluence (data not shown), was accompanied by a marked decrease in Myt1 levels. During the transition from G0 into the cell cycle Myt1 protein was first detected during early S-phase, analogous to Wee1 when overexpressed in the fission yeast S. similar to the kinetics previously reported for its primary pombe. Although a homologue of Myt1 has not yet been substrate Cdc2 (Fig. 3a, lower panel) (Pagano et al., 1993). As described in S. pombe, Thr14 is conserved in S. pombe Cdc2 expected, a shift to a faster mobility form of Cdc2, due to and phosphorylation of Thr14 is observed under some Thr14/Tyr15 dephosphorylation during M-phase (Solomon et circumstances (Den Haese et al., 1995). Wild-type and al., 1992), was observed at 27-30 hours following release from catalytically inactive Myt1 were cloned into a fission yeast serum starvation. Myt1 protein was readily detectable at this expression vector (pSLF273) that uses the mid-strength nmt time implying that degradation of Myt1 does not precede the promoter (Forsburg and Sherman, 1997). The resulting dephosphorylation and therefore the activation of Cdc2. plasmids and vector alone were used to transform a wild-type Next we synchronized U2-OS cells at the beginning of S- S. pombe strain 1058, and the effect of Myt1 expression phase by treatment with thymidine, then washed, released following thiamine depletion, which derepresses transcription and promptly blocked the cells in metaphase by addition of Regulation of Myt1 3365

Fig. 2. Overexpression of Myt1 perturbs normal S. pombe cell cycle progression. Photomicrographs of DAPI stained S. pombe cells transformed with vector alone (pSLF273), or Myt1 driven by the nmt promoter, either under inducing conditions (minus thiamine) or repressed conditions (plus thiamine), subsequently grown in medium with and without thiamine. the microtubule-depolymerizing drug nocodazole. Cells Localization of MPM-2 epitopes in Myt1 were subsequently released from the drug-induced block and The monoclonal antibody MPM-2, which recognizes mitotic samples taken at time intervals for flow cytometric analysis phosphoepitopes, has been an extremely useful tool for (Fig. 3b) and, in parallel, a portion was retained for the dissecting the function of mitotic phosphorylation of many preparation of cell extracts. Western blotting analysis proteins (Davis et al., 1983). To determine whether mitotic indicated that Myt1 protein levels did not fluctuate hyperphosphorylated Myt1Hu contains MPM-2 epitopes, dramatically during the cell cycle in proliferating U2-OS transfected Myc-tagged Myt1Hu was immunoprecipitated cells (Fig. 3b). A small decrease in the level of the lower with αMyc antibody from mitotic cellular extracts, prepared Myt1 protein band was observed in G2/M phase extracts, from 293T cells arrested in metaphase with nocodazole, and although not dramatic and may indeed result from a analyzed by western blotting with the MPM-2 antibody. The reduction in mobility on SDS-PAGE following full length protein was recognized by the MPM-2 antibody hyperphosphorylation in mitosis (see below). Similar results were also obtained when U2-OS cells and HeLa cells that were synchronized at the beginning of S-phase by a double thymidine block and released, enter mitosis (data not shown). These data suggest that Myt1 is a proliferation marker that is downregulated upon quiescence, consistent with a role in the regulation of the G2/M phase transition, but is not regulated at the protein level in proliferating cells. Although western blotting did not detect a significant decrease in Myt1 as cells enter mitosis, the protein had a dramatically slower mobility on SDS-PAGE in extracts prepared from cells blocked in metaphase (Fig. 3b). Previous studies have determined that this shift is due to hyperphosphorylation at mitosis (Mueller et al., 1995; Liu et al., 1997; Booher et al., 1997), which is coincident with Myt1 inactivation. Hence, we next examined whether phosphorylation of Myt1 was of importance for the regulation of the Cdc2 inhibitory activity of Myt1.

Fig. 3. Myt1 is downregulated in quiescent human fibroblasts. (a) G0 HSF8 cells were stimulated to reenter the cell cycle and sampled at the indicated times. Cell lysates were subjected to SDS-PAGE, transferred to Immobilon-P and then blotted with αMyt1 (upper panel) and αCdc2 (lower panel) antibodies, as marked. Two Cdc2 bands are arrowed which represent different phosphorylated forms of the kinase. Analysis of DNA content by flow cytometry of samples is shown below. The percentage of cells in each specific phase of the cell cycle in each sample are indicated. (b) U2-OS cells were released from a metaphase arrest and sampled at indicated times. Cell lysates were subjected to SDS-PAGE, transferred to Immobilon- P and then blotted with αMyt1. Hyperphosphorylated mitotic Myt1 is indicated. Analysis of DNA content by flow cytometry of samples is shown below. The percentage of cells in each specific phase of the cell cycle in each sample are indicated. 3366 N. J. Wells and others

truncation of the C-terminal domain at residue Gly408 (∆C- Myt1), up to but not including the putative transmembrane domain, abolished reactivity with the MPM-2 antibody (Fig. 4b, left panel). The blot was stripped and reprobed with αMyc antibody to demonstrate that all the Myc-tagged Myt1 fusion proteins were immunoprecipitated (Fig. 4b, right panel). Liu et al. (1997) have previously demonstrated that truncation of the C-terminal 98 residues, leaving the putative transmembrane domain intact, has no effect on subcellular localization of Myt1, and thus the ∆C-Myt1 protein should have been present in the same cellular environment as wild- type Myt1, and was therefore in a position to be phosphorylated. In conclusion, the major MPM-2 reactivity is localized to the C-terminal domain of Myt1, which contains 5 putative CDK phosphorylation sites and therefore putative MPM-2 epitopes. Consistent with the deletion studies, mutation of all 5 CDK consensus phosphoacceptor residues, Thr412, Ser416, Ser441, Thr455 and Thr461, prevented recognition of Myt1 by the MPM-2 antibody (data not shown). To determine which if any of these CDK consensus phosphorylation sites, within the C- terminal domain, are targets for CDK phosphorylation, a GST fusion with the C-terminal domain of Myt1 was constructed (residues Ala401-COOH) (Fig. 4a). An in vitro kinase assay demonstrated that the C-terminal domain is a good substrate for Cdc2/cyclin B1 (Fig. 4c). Interestingly phosphorylation of the C-terminal domain dramatically reduced its SDS-PAGE mobility (Fig. 4c) as observed for the wild-type protein during mitosis (Fig. 3b) (Mueller et al., 1995; Booher et al., 1997). Furthermore, a range of decreased electrophoretic mobilities of GST-COOH was observed by SDS-PAGE following phosphorylation, indicative of phosphorylation at multiple sites. Pin1 interacts with Myt1 in a phosphorylation dependent manner Fig. 4. Localization of MPM-2 epitopes in Myt1. (a) Diagrammatic representation of the Myt1 truncations used in this study. * denotes Pin1, a petidyl-prolyl isomerase (PPIase), may regulate the putative CDK consensus sites within the C-terminal domain (only entry into and progression through mitosis (Lu et al., 1996). shown on the GST-COOH fusion protein for simplicity). The black Structural analysis of the PPIase active site led to the section represents the transmembrane domain (TMD). Numbers refer hypothesis that Pin1 recognizes its substrates with acidic to amino acids. Note the N-terminal 6Myc tag is not shown. (b) residues prior to the isomerized N-terminal proline peptide Immunoprecipitates of the Myt1 deletion constructs described in (a), bond (Ranganathan et al., 1997). Recently, Yaffe et al. (1997) from metaphase 293T cell extracts, were subjected to SDS-PAGE demonstrated that indeed Pin1 preferentially binds to and then transferred to Immobilon-P before blotting with αMPM-2 and α isomerizes proline bonds immediately preceded by a Myc antibodies as marked. (c) In vitro phosphorylation of GST- phospho-Thr or phospho-Ser, similar to the minimal MPM-2 COOH described in (a) by purified baculovirus-expressed Cdc2/cyclin B1. GST-COOH or GST alone was incubated with [γ- epitope. Pin1 may therefore regulate mitosis by 32P]ATP in the presence or absence of Cdc2/cyclin B1, subjected to phosphorylation specific isomerization of the substrates of SDS-PAGE and visualized by autoradiography (left), or in an the MPM-2 kinases. Subsequent work has shown that this independent experiment with unlabelled ATP, Coomassie blue binding specificity is largely a property of the Pin1 WW staining (right). domain (Lu et al., 1999). To investigate whether Myt1 and Pin1 interact, 293T cells were transiently cotransfected with HA-tagged Pin1 and Myc- (Fig. 4b, left panel), consistent with the results for Xenopus tagged Myt1 expression constructs, and cell extracts were Myt1 (Mueller et al., 1995). To determine the region(s) in prepared. Immunoprecipitation of either Myt1 or Pin1, via the which the epitope(s) are localized, we prepared Myt1 epitope tags, and subsequent western analysis demonstrated expression constructs with either N- or C-terminal deletions, that Pin1 and Myt1 did indeed interact. Immunoprecipitation or both (Fig. 4a), and expressed these transiently in 293T cells. of Myt1 and the subsequent detection of Proteins were immunoprecipitated with αMyc from mitotic coimmunoprecipitated Pin1 is shown in Fig. 5a. Analysis of cell extracts, and blotted with the MPM-2 antibody. Deletion Pin1 coexpressed with either ∆N-Myt1 and ∆C-Myt1 (Fig. 4a) of the N terminus (∆N-Myt1) up to and including residue demonstrated that this interaction was dependent on the C- Pro102 had little effect on the MPM-2 reactivity. In contrast, terminal domain of Myt1 (Fig. 5a). This suggests that the Regulation of Myt1 3367

MPM-2 epitopes in the C-terminal domain of Myt1 participate results demonstrate that Pin1 interacts directly with the C- in this interaction. terminal domain of Myt1 in a phosphorylation-dependent Two approaches were pursued to examine the dependence manner. However, we have been unable to detect any of the interaction on phosphorylation. First, mutation of the five modulation of in vitro Myt1 catalytic activity through its consensus CDK phosphorylation sites, Thr412, Ser416, association with Pin1 (T. Tokusumi and N. Watanabe, Ser441, Thr455 and Thr461 (Ser/Thr to Ala), in the Myt1 C unpublished observations). terminus (Fig. 4a) significantly reduced the coimmunoprecipitation of wild-type Pin1 with Myt1 (data not The C-terminal domain is required for the biological shown). However, mutation of these phosphoacceptor sites did activity of Myt1 not abrogate its ability to induce a G2/M arrest in U2-OS cells The fact that Myt1 mitotic hyperphosphorylation is coincident (data not shown). Second, utilizing the GST-COOH fusion with its inactivation, coupled with our localization of the protein (Fig. 4a and 4c) in a pull down assay, we demonstrated mitotic MPM-2 epitopes to the C-terminal domain, suggests that Pin1 only interacted with the C-terminal domain following that phosphorylation of these sites could regulate the function Cdc2/cyclin B1 phosphorylation (Fig. 5b). Together these of the C-terminal domain. To determine whether this domain is required for Myt1 biological activity in mammalian cells, ∆C-Myt1 (Fig. 4a) and a catalytically inactive variant of ∆C- Myt1 (Asp251Ala), were transfected into U2-OS cells and the effect on cell cycle progression was examined by flow cytometric analysis. Surprisingly, transient transfection studies in U2-OS cells demonstrated that ∆C-Myt1 (Fig. 6a) or ∆C- Myt1 (Asp251Ala) (data not shown) were unable to induce the G2/M arrest phenotype observed for the wild-type control. Western blotting analysis indicated that the truncation mutant was expressed as well or better than wild-type Myt1 (Fig. 6b). Next we examined the ability of ∆C-Myt1 to phosphorylate Cdc2 compared to that of wild-type Myt1. We carried out in vitro kinase assays on αMyc-immunoprecipitates of Myc- tagged wild-type Myt1 and ∆C-Myt1 from asynchronous transfected 293-T cells cell extracts using the inactive kinase Cdc2(KR)/cyclin B1 as a substrate. Consistent with the observation that ∆C-Myt1 does not cause a G2/M arrest in U2- OS cells, deletion of the C-terminal domain significantly reduced Myt1 phosphorylation of Cdc2 (Fig. 6c). Interestingly, ∆C-Myt1 underwent autophosphorylation to the same extent as full length Myt1 (data not shown), and hence is still enzymatically active regardless of the C-terminal truncation. In contrast, as expected the Myt1 (Asp251Ala) mutant lacked both Cdc2 phosphorylating activity and autophosphorylating activity. Analysis by western blotting indicated that equal amounts of the Myt1 proteins, utilized in the kinase assays, were present in the cell extracts (Fig. 6c, right panel). These results imply that the C-terminal region plays some role in the interaction of Myt1 with its substrate Cdc2/cyclin B1. To determine whether the C-terminal domain is required for the recognition and recruitment of its substrate, Cdc2/cyclin B1, we immunoprecipitated wild-type Myt1 and ∆C-Myt1 from 293T cell extracts and resolved the Fig. 5. In vivo and in vitro interaction of Myt1 and Pin1. (a) Myt1 coimmunoprecipitating proteins by SDS-PAGE. Western (6Myc-Myt1 deletion series) and Pin1 (HA-Pin1) were cotransfected blotting analysis with αCdc2 antibodies demonstrated that together or with vector alone controls into 293T cells as indicated. endogenous Cdc2 coimmunoprecipitated with wild-type Myt1 Immunoprecipitations were performed with αMyc antibodies and the but not with ∆C-Myt1 (Fig. 6d). The blot was stripped and coimmunoprecipitated Pin1 protein detected by western blotting with reprobed with αMyc antibody to demonstrate that ∆C-Myt1 αHA antibodies (top panel). Samples of the whole cell lysates was indeed immunoprecipitated. Interestingly, the majority of (WCL) are also included. The expression and immunoprecipitation the associated Cdc2 was in the slowest mobility form, of the Myc tagged Myt1 proteins is shown in the lower panel after representing the doubly-phosphorylated and hence inactive reblotting the membrane with αMyc antibodies. (b) GST pull-down assay. GST-COOH (Fig. 4a) or GST alone was incubated with ATP Cdc2. Previous studies have demonstrated that Myt1 will only in the presence or absence of Cdc2/cyclin B1, bound to glutathione recognize and therefore phosphorylate its substrate, Cdc2, in a agarose, washed and subsequently incubated with Pin1 before cyclin B1-dependent manner (Booher et al., 1997). Since we washing once more. Bound Pin1 was resolved by SDS-PAGE and observed only doubly-phosphorylated Cdc2 in association with detected by western blotting with αPin1 antibody. Myt1 (Fig. 6d), we deduce that Cdc2/cyclin B complexes were 3368 N. J. Wells and others

Fig. 6. Myt1 C-terminal domain is required for biological activity. (a) Flow cytometric analyses of GFP-negative and -positive U2-OS cells cotransfected with a Myt1 expression construct (20 µg) or ∆C- Myt1 and the EGFPF construct (5 µg). The propidium iodide signal was used as a measure of DNA content to determine the cell cycle profiles (G1, S and G2/M). The DNA histograms from each assay 4 contain data from 10 cells. The percentage of cells in G2/M phases of the cell cycle are shown. (b) Equal amounts of whole cell lysate from cells analysed in (a) were subjected to SDS-PAGE and western blotting with the αMyc antibodies. (c) Myc tagged Myt1, Myt1-D/A (Asp251Ala) and ∆C-Myt1 were immunoprecipitated from 293T cell extracts following transfection, and utilized in an in vitro kinase assay using Cdc2(KR)/Cyclin B as the substrate and incubated with [γ32P]ATP. The reaction was resolved on SDS-PAGE and then subjected to autoradiography (left panel). Equal amounts of whole cell lysates (WCL) utilized for immunoprecipitations and subseqent kinase assays were resolved on SDS-PAGE and western blotting analysis with αMyc antibodies performed (right panel). (d) Following 293T cell transfection with vector alone, Myc tagged ∆C-Myt1, wild-type Myt1 and Myt1-D/A (Asp251Ala), cell extracts were prepared and then subsequent immunoprecipitations with αMyc antisera were carried out. Western blotting analysis of the αMyc immunoprecipitations (αMyc.IP) and whole cell lysates (WCL) are shown: αCdc2 top panel (the three forms of cyclin B associated Cdc2 are indicated), αMyc bottom panel.

coimmunoprecipitated with Myt1. In contrast, both doubly- phosphorylated, inactive Cdc2 and hypophosphorylated, active Cdc2, were coimmunoprecipitated with the catalytically inactive Myt1 (Asp251Ala) protein (Fig. 6d); however, the level of the hypophosphorylated, active Cdc2 brought down was proportionately much less than the level of this form in the whole cell lysate. The presence of some doubly- phosphorylated Cdc2 associated with catalytically inactive Myt1 (Asp251Ala) could be due to its phosphorylation by endogenous Myt1.

DISCUSSION

We have demonstrated that inappropriate expression/activity of Myt1 prevents cells of both lower and higher eukaryotes from completing cell division after DNA replication. This is consistent with the fact that overexpression of a dominant negative Cdc2, to date the unique substrate of Myt1, causes cells to arrest at the G2 to M transition (van den Heuvel and Harlow, 1993). Our studies also indicate that the C-terminal domain is essential for the biological activity of Myt1, as indicated by its requirement for Myt1 to cause a G2/M arrest, and its role in recruitment and phosphorylation of Cdc2/cyclin B1 in vitro. Expression studies in mammalian cells of a non- phosphorylatable Cdc2 (Thr14/Ala-Tyr15/Phe) (Cdc2AF) indicate that inhibitory phosphorylation is not solely responsible for repressing premature mitosis (Heald et al., 1993; Jin et al., 1996). The initiation of mitosis is accompanied by the dephosphorylation/activation of Cdc2 and translocation to the nucleus (Pines and Hunter, 1991, 1994). Heald et al. (1993) demonstrated that dephosphorylated/active Cdc2, which is restricted to the cytoplasm, is unable to catalyze entry into M phase. Regulated subcellular localization of Cdc2 may also explain the poor efficiency with which expression of Cdc2AF induces premature entry into mitosis (Heald et al., Regulation of Myt1 3369

1993; Jin et al., 1996, 1998). This is consistent with our metaphase by nocodazole and subsequently treated with DNA observation that overexpression of a catalytically inactive Myt1 damaging agents, lose Cdc2/cyclin B activity concomitant with (Asp251Ala) mutant was also able to delay cell cycle the appearance of phosphorylation of Thr14 and Tyr15 on progression in mammalian cells, which we suggest is due to its Cdc2 (Poon et al., 1997). This implies that a Thr14 kinase is ability to bind Cdc2/cyclin B1 and sequester this complex in activated or at least derepressed during DNA damage in the the cytoplasm. Sequestration of the mitotic kinase within the metaphase arrest; this Thr14 kinase is probably Myt1, which cytoplasmic compartment, thereby preventing its translocation is the major Thr14 kinase in Xenopus eggs (Mueller et al., to the nucleus, would effectively inhibit mitotic entry. Thus, 1995). In contrast to Myt1Hu, Wee1Hu protein levels are Myt1 can inhibit Cdc2 and hence entry into mitosis by two decreased at M/G1 phases, highlighting a further difference in independent mechanisms. First, by binding and sequestration the regulation of the two higher eukaryotic Cdc2 inhibitory of Cdc2/cyclin B1 from the nucleus, and second following kinases (Watanabe et al., 1995; McGowan and Russell, 1995). recruitment of Cdc2/cyclin B1, by inhibitory phosphorylation Myt1 inactivation is coincident with its mitotic of Thr14 and Tyr15. This model has received independent hyperphosphorylation (Mueller et al., 1995; Booher et al., support from Liu et al. (1999), who have also shown that the 1997). Utilizing deletion mutants we have localized the MPM- C-terminal domain of Myt1 interacts with Cdc2/cyclin B1 via 2 epitopes, and hence mitotic specific phosphorylation sites an RXL cyclin-binding motif, RNL, located within the C- within Myt1 to the C-terminal domain, which contains 5 terminal 63 residues of Myt1, and that this interaction is putative CDK Ser/Thr-Pro phosphorylation sites. We found essential for kinase-inactive Myt1 to arrest mammalian cells in that the Myt1 C-terminal domain is a good substrate for G2. In addition, Liu et al. have also shown that the Myt1 C- Cdc2/cyclin B1 complex in vitro, and that Cdc2/cyclin B1 terminal domain sequesters cyclin B1 in the cytoplasm of cells phosphorylation also led to a dramatic reduction in mobility on treated with leptomycin B, where it normally accumulates in SDS-PAGE, as is observed for Myt1 in mitotic cells (Mueller the nucleus, thus providing compelling evidence that Myt1 can et al., 1995; Booher et al., 1997). Cell cycle phase specific negatively regulate Cdc2/cyclin B1 by preventing its entry into phosphorylation of Myt1 at the N terminus has also been the nucleus. predicted, based on the observation that an αMyt1 N-terminal In contrast to its inhibitory effect in human cells, peptide antibody is able to recognize interphase but not mitotic catalytically-inactive Myt1Hu did not induce a cell cycle arrest Myt1 (Booher et al., 1997). The N terminus also contains in S. pombe. This difference may be due to differing expression consensus CDK phosphorylation sites, but our results suggest levels or a lower affinity of Myt1Hu for S. pombe Cdc2/Cdc13 that the MPM-2 epitope monoclonal antibody is unable to (Cdc13 is the S. pombe cyclin B1 homologue). Transformation recognize these phosphorylation sites, if they are indeed of Myt1Hu, under the full strength nmt promoter, into a modified, and that Pin1 is also unable to interact with this humanized Cdc2 S. pombe strain (h−, leu1-32, his3-237, domain. cdc2::CDC2Hu) under repressed conditions prevented colony Pin1 associates with epitopes that overlap with those of the formation, whereas colonies were obtained following a similar mitotic phosphoepitope specific MPM-2 monoclonal antibody transformation of a wild-type S. pombe strain (N. J. Wells, (Yaffe et al., 1997). Consistent with that study, we have unpublished observations). Thus, the low level of Myt1Hu demonstrated that Pin1 interacts with Myt1 in vivo and in vitro expressed in repressed cells was sufficient to arrest S. pombe in a phosphorylation dependent manner. Whilst this work was cells relying on human Cdc2, but insufficient to arrest cells in preparation Shen et al. (1998) also demonstrated that the with endogenous Cdc2. This suggests that Myt1Hu does have mitotic hyperphosphorylated form of Myt1 was specifically a higher affinity for human Cdc2 than S. pombe Cdc2. This isolated in a Pin1-GST pull down assay. We extended this could be because human Cdc2 is intrinsically a better substrate, preliminary observation by demonstrating that it is a direct, but it is also possible that the S. pombe Cdc13 cyclin has a phosphorylation-dependent interaction, and requires one or lower affinity for the RNL motif in Myt1 than human cyclin more of the 5 CDK consensus sites in the C-terminal domain B1. Furthermore, S. pombe Cdc2/Cdc13 is reportedly restricted of Myt1. Interestingly, Pin1 also interacts with two other Cdc2 to the nuclear compartment (Alfa et al., 1989), and this may in regulatory proteins, Wee1 and Cdc25C (Crenshaw et al., 1998; part protect the Cdc2 kinase from sequestration by Myt1 in the Shen et al., 1998). As the phosphorylation of Thr14/Tyr15, and cytoplasm, although a more recent study indicates that the hence the activity of Cdc2, is tightly regulated by feedback translocation of Cdc2/Cdc13 into the nuclear compartment loops (reviewed by Coleman and Dunphy, 1994), the ability of may even occur in S. pombe (Audit et al., 1996). We have been Pin1 to interact with Cdc25, Wee1 and Myt1 provides the basis unable to suppress the wee1-50 mutant phenotype by for modulation of these feedback pathways. To date direct expression of Myt1Hu from the nmt promoter (N. J. Wells, changes in enzymatic activity due to conformational control unpublished observations), which may also in part be catalyzed by the PPIase domain of Pin1 are still debatable. We accounted for by different subcellular locations of Myt1 and examined the effect of Pin1 association with Myt1 in vitro and Wee1, which is nuclear. did not detect a significant change in the kinase activity of Western blotting analyses indicate that Myt1 protein is solubilized Myt1 in an immunoprecipitate (N.T. and N.W., dramatically downregulated upon cell contact inhibition or unpublished observations). Future studies are needed with serum starvation in normal human fibroblasts. We did not membrane-associated Myt1, where it may adopt a different observe a significant decrease in expression following transit conformation, to study the regulatory role of modifications through mitosis in either U2-OS or HeLa cells. This is including phosphorylation and Pin1 interaction on enzyme consistent with the observation that when cells are arrested in activity. metaphase, by treatment with nocodazole, Myt1 protein is still Initial fractionation studies in Xenopus egg and HeLa cell present (Fig. 3) (Booher et al., 1997). Indeed, cells blocked in extracts showed that the Cdc2 Thr14 kinase was membrane 3370 N. J. Wells and others associated and that its membrane association was not disrupted Audit, M., Barbier, M., Soyer-Gobillard, M. O., Albert, M., Geraud, M. by high salt treatment (Kornbluth et al., 1994; Atherton et al., L., Nicolas, G. and Lenaers, G. (1996). Cyclin B (p56cdc13) localization 1994). Subsequently, a membrane-targeting domain was in the yeast Schizosaccharomyces pombe: an ultrastructural and immunocytochemical study. Biol. Cell. 86, 1-10. identified in the Myt1 sequence downstream of the catalytic Booher, R. N., Holman, P. S. and Fattaey, A. (1997). Human myt1 is a cell domain, and was shown by immunofluorescence staining to be cycle-regulated kinase that inhibits cdc2 but not cdk2 activity. J. Biol. Chem. required for the localization of Myt1 to the ER and Golgi 272, 22300-22306. complex (Liu et al., 1997). From these data it was predicted Coleman, T. R. and Dunphy, W. G. (1994). Cdc2 regulatory factors. Curr. Opin. Cell Biol. 6, 877-882. that the kinase homology domain is oriented toward the Crenshaw, D. G., Yang, J., Means, A. R. and Kornbluth, S. (1998). The cytosolic face of these membrane compartments, but it was mitotic peptidyl-prolyl isomerase, Pin1, interacts with Cdc25 and Plx1. unclear whether the C-terminal tail is in the lumen of the ER EMBO J. 17, 1315-1327. (i.e. a type II transmembrane protein), or whether it is also in Davis, F. M., Tsao, T. Y., Fowler, S. K. and Rao, P. N. (1983). Monoclonal the cytosol. From our work showing that the Myt1 C-terminal antibodies to mitotic cells. Proc. Nat. Acad. Sci. USA 80, 2926-2930. Den Haese, G. J., Walworth, N., Carr, A. M. and Gould, K. L. (1995). The domain binds Cdc2/cyclin B1 in interphase and is Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2. phosphorylated in mitosis, we infer that the Myt1 C-terminal Mol. Biol. Cell 6, 371-385. domain is exposed in the cytoplasm, where it is able to recruit Forsburg, S. L. and Sherman, D. A. (1997). General purpose tagging vectors Cdc2/cyclin B1, an event necessary for phosphorylation of for fission yeast. 191, 191-195. Thr14 and Tyr15. Hagting, A., Karlsson, C., Clute, P., Jackman, M. and Pines, J. (1998). MPF localization is controlled by nuclear export. EMBO J. 17, 4127-4138. A previous study indicates that phosphorylation of Myt1 by Heald, R., McLoughlin, M. and McKeon, F. (1993). Human wee1 maintains Cdc2 in vitro does not inhibit its Thr14/Tyr15 kinase activity mitotic timing by protecting the nucleus from cytoplasmically activated (Booher et al., 1997). Perhaps some regulatory factor that Cdc2 kinase. Cell 74, 463-474. associates with phosphorylated Myt1 is absent and/or another Heintz, N., Sive, H. L. and Roeder, R. G. (1983). Regulation of human histone : kinetics of accumulation and changes in the rate protein kinase(s) is also required for inhibition. One possibility of synthesis and in the half-lives of individual histone mRNAs during the is that mitotic phosphorylation of the Myt1 C-terminal domain HeLa cell cycle. Mol. Cell. Biol. 3, 539-550. could decrease the binding of cyclin B1/Cdc2, and recent Igarashi, M., Nagata, A., Jinno, S., Suto, K. and Okayama, H. (1991). evidence suggests that this is indeed the case (Liu et al., 1999). Wee1(+)-like gene in human cells. Nature 353, 80-83. Palmer et al. (1998) have recently demonstrated that p90Rsk can Janknecht, R. (1996). Analysis of the ERK-stimulated ETS ER81. Mol. Cell. Biol. 16, 1550-1556. also bind and to phosphorylate the Myt1 C-terminal domain. Jiang, W. and Hunter, T. (1998). Analysis of cell-cycle profiles in transfected Their data suggest that phosphorylation of the C-terminal cells using a membrane-targeted GFP. Biotechniques 24, 349-354. domain by p90Rsk inhibits the ability of Myt1 to phosphorylate Jin, P., Gu, Y. and Morgan, D. O. (1996). Role of inhibitory CDC2 and inhibit Cdc2. It will be interesting to examine whether phosphorylation in radiation-induced G2 arrest in human cells. J. Cell Biol. Rsk 134, 963-970. phosphorylation of the C-terminal domain by p90 might Jin, P., Hardy, S. and Morgan, D. O. (1998). Nuclear localization of also inhibit Myt1 activity by decreasing the affinity of the Myt1 cyclin B1 controls mitotic entry after DNA damage. J. Cell Biol. 141, 875- C-terminal domain for Cdc2/cyclin B complexes. This study 885. also demonstrates that the C-terminal domain of Myt1 is Kanemitsu, M. Y., Jiang, W. and Eckhart, W. (1998). Cdc2-mediated sufficient for interaction with Cdc2. 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