Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

The retinoblastoma protein physically associates with the human cdc2 kinase.

The protein product (pRB) of the retinoblastoma susceptibility gene functions as a negative regulator of cell proliferation, and its activity appears to be modulated by phosphorylation. Using a new panel of anti-human pRB monoclonal antibodies, we have investigated the biochemical properties of this protein. These antibodies have allowed us to detect a pRB-associated kinase that has been identified as the cell cycle-regulating kinase p34cdc2 or a closely related enzyme. Since this associated kinase phosphorylates pRB at most of the sites used in vivo, these results suggest that this kinase is one of the major regulators of pRB. The associated kinase activity follows the pattern of phosphorylation seen for pRB in vivo. The associated kinase activity is not seen in the G1 phase but appears in the S phase, and the levels continue to increase throughout the remainder of the cell cycle.     

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products.

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.     

Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle.

The activity of p34cdc2 kinase is regulated in the phases of vertebrate cell cycle by mechanisms of phosphorylation and dephosphorylation. In this paper, we demonstrate that casein kinase II (CKII) phosphorylates p34cdc2 in vivo and in vitro at Ser39 during the G1 phase of HeLa cell division cycle. Human p34cdc2 shows a typical phosphorylation sequence motif site for CKII at Ser39 (ES39EEE). In our experiments, either p34cdc2 expressed and purified from bacteria or p34cdc2 immunoprecipitated from HeLa cells enriched in G1 by elutriation were substrates for in vitro phosphorylation by CKII. Phosphoamino acid analysis, N-chlorosuccinimide mapping, and two-dimensional tryptic mapping of p34cdc2 phosphorylated in vitro were performed to determine the phosphorylation site. A synthetic peptide spanning residues 33-50 of human p34cdc2, including the CKII site, was used to map the site. In addition, phosphorylation at Ser39 also occurs in vivo, since p34cdc2 is phosphorylated during G1 on serine, and its two-dimensional tryptic map shows two phosphopeptides that comigrate exactly with the synthetic peptides used as standard.     

High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase

All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle.     

Transforming growth factor beta 1 (TGF beta 1) reduces cellular levels of p34cdc2, and this effect is abrogated by adenovirus independently of the E1A-associated pRB binding activity.

We have used E1A probes to study the roles of the p34cdc2 kinase and the retinoblastoma tumor susceptibility gene product (pRB) in transforming growth factor beta 1 (TGF beta 1)-mediated growth suppression in mink lung epithelial (Mv1Lu) cells. In agreement with previous reports, we see a decline in p34cdc2 kinase activity and a loss of pRB phosphorylation after TGF beta 1 treatment. We report here that TGF beta 1 induces not only a change in p34cdc2 kinase activity but a strong repression of p34cdc2 synthesis. Loss of p34cdc2 kinase activity is not seen until the steady-state level of p34cdc2 declines, suggesting that the intra-cellular signals induced by TGF beta 1 affect p34cdc2 at the level of expression, rather than by altering the posttranslational modifications of p34cdc2 that regulate its kinase activity. Infection with adenovirus expressing either wild-type E1A or a mutant E1A (pm928) defective for pRB binding alleviated TGF beta 1-mediated suppression of DNA synthesis, indicating that E1A does not need to bind pRB physically to keep cell growth-suppressing functions from being activated by TGF beta 1. The E1A.928 mutant virus is able to maintain p34cdc2 expression and kinase activity, as well as pRB phosphorylation in the presence of TGF beta 1, which may account for its ability to maintain cell cycle activity without directly sequestering pRB. Overall our results suggest that TGF beta 1 acts by signaling changes at the level of control of G1 gene expression, not at the level of posttranslational modification of p34cdc2 or its substrates.     

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.     

Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis.

In human epidermal carcinoma A431 cells, the beta subunit of casein kinase II is phosphorylated at an autophosphorylation site and at serine 209 which can be phosphorylated in vitro by p34cdc2 (Litchfield, D. W., Lozeman, F. J., Cicirelli, M. F., Harrylock, M., Ericsson, L. H., Piening, C. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 20380-20389). Given the importance of p34cdc2 in the regulation of cell cycle events, we were interested in examining the phosphorylation of casein kinase II during different stages of the cell cycle. In this study it is demonstrated that the extent of phosphorylation of serine 209 in the beta subunit is significantly increased relative to phosphorylation of the autophosphorylation site when chicken bursal lymphoma BK3A cells are arrested at mitosis by nocodazole treatment. This result suggests that serine 209 is a likely physiological target for p34cdc2. In addition, the alpha subunit of casein kinase II also undergoes dramatic phosphorylation with an associated alteration in its electrophoretic mobility when BK3A cells or human Jurkat cells are arrested with nocodazole. Phosphopeptide mapping studies indicate that p34cdc2 can phosphorylate in vitro the same peptides on the alpha subunit that are phosphorylated in cells arrested at mitosis. These phosphorylation sites were localized to serine and threonine residues in the carboxyl-terminal domain of alpha. Taken together, the results of this study indicate that casein kinase II is a probable physiological substrate for p34cdc2 and suggest that its functional properties could be affected in a cell cycle-dependent manner.     

A pre-start checkpoint preventing mitosis in fission yeast acts independently of p34cdc2 tyrosine phosphorylation.

We have monitored the tyrosine (Y15) phosphorylated and dephosphorylated forms of p34cdc2 from Schizosaccharomyces pombe as cells proceed through the cell cycle. Y15 is dephosphorylated in G1 before start and becomes phosphorylated only after cells pass start and enter late G1. This transition is associated with a switch from one checkpoint which restrains mitosis in pre-start G1, by a mechanism independent from Y15 phosphorylation, to a second checkpoint acting post-start during late G1 and S phase operating through Y15 phosphorylation. The pre-start checkpoint may act by preventing formation of the p34cdc2/p56cdc13 complex. The complex between Y15-phosphorylated p34cdc2 and p56cdc13 accumulates during S phase and G2, but the level generated is not solely dependent on the amount of p34cdc2 and p56cdc13 present in the cell. The extent of p56cdc13 breakdown at the end of mitosis may be determined by the amount complexed with p34cdc2. We have also shown that an insoluble form of p34cdc2 is associated with the progression of the cell through late G1 into S phase.     

Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2.

The carboxyl-terminal regions of neurofilament high (NF-H) and middle (NF-M) molecular weight proteins have been suggested to be phosphorylated in vivo by a p34cdc2-like protein kinase, on the basis of the in vivo phosphorylation site motif and in vitro phosphorylation of the proteins by p34cdc2 kinase (Hisanaga, S.I., Kusubata, M., Okumura, E. and Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803). A novel proline-directed protein kinase previously identified and purified from bovine brain has been found in this study to phosphorylate NF-H and NF-M at sites identical to those phosphorylated by HeLa cell p34cdc2 kinase. The proline-directed kinase is composed of a 33-kDa and a 25-kDa subunit. The 33-kDa kinase subunit was partially sequenced, and degenerate oligonucleotide primers corresponding to the amino acid sequence information were used to clone the subunit by polymerase chain reaction (PCR). Two overlapping PCR products comprised a complete open reading frame of 292 amino acids. The sequence contains all features of a protein kinase, suggesting that the 33-kDa peptide represents the catalytic subunit of the kinase. The 33-kDa subunit shows high and approximately equal homology to human p34cdc2 and human cdk2, with about 58 and 59% amino acid identity, respectively. These results suggest that the brain kinase represents a new category of the cdc2 family, and that some members of the cdc2 kinase family may have major functions unrelated to cell cycle control.     

Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.

With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.     

Cell cycle regulation of vertebrate p34cdc2 activity: identification of Thr161 as an essential in vivo phosphorylation site.

The protein kinase p34cdc2 is a key regulator of the cell cycle in all eukaryotes. Its activity is controlled by cell cycle-dependent interactions with other proteins, notably cyclins, and by changes in its phosphorylation state. Two inhibitory phosphorylation sites in chicken p34cdc2 have previously been mapped to threonine 14 and tyrosine 15. Here we describe the identification of threonine 161 as an additional in vivo phosphorylation site in vertebrate p34cdc2. Phosphorylation of this site is cell cycle dependent and likely to be required for p34cdc2 activity.     

The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells.

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.     

Cyclin-dependent kinases phosphorylate the adenovirus E1A protein, enhancing its ability to bind pRb and disrupt pRb-E2F complexes.

The adenovirus E1A protein of 243 amino acids has been shown to affect a variety of cellular functions, most notably the immortalization of primary cells and the promotion of quiescent cells into S phase. The activity of E1A is derived, in part, from its association with various cellular proteins, many of which play important roles in regulating cell cycle progression. E1A is known to have multiple sites of phosphorylation. It has been suggested that cell cycle-dependent phosphorylation may also control some of E1A's functions. We find now that immune complexes of cyclin-dependent kinases such as cdk4, cdk2, and cdc2 are all capable of phosphorylating E1A in vitro. Additionally, the sites on E1A phosphorylated by these kinases in vitro are similar to the E1A sites phosphorylated in vivo. We have also found that a phosphorylated E1A is far more efficient than an unphosphorylated E1A in associating with pRB and in disrupting E2F/DP-pRB complexes as well. On the basis of our findings and the differences in timing and expression levels of the various cyclins regulating cdks, we suggest that E1A functions at different control points in the cell cycle and that phosphorylation controls, to some extent, its biological functions.     

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.     

Mutations at phosphorylation sites of Xenopus microtubule-associated protein 4 affect its microtubule-binding ability and chromosome movement during mitosis

Microtubule-associated proteins (MAPs) bind to and stabilize microtubules (MTs) both in vitro and in vivo and are thought to regulate MT dynamics during the cell cycle. It is known that p220, a major MAP of Xenopus, is phosphorylated by p34(cdc2) kinase as well as MAP kinase in mitotic cells, and that the phosphorylated p220 loses its MT-binding and -stabilizing abilities in vitro. We cloned a full-length cDNA encoding p220, which identified p220 as a Xenopus homologue of MAP4 (XMAP4). To examine the physiological relevance of XMAP4 phosphorylation in vivo, Xenopus A6 cells were transfected with cDNAs encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein. Mutations of serine and threonine residues at p34(cdc2) kinase-specific phosphorylation sites to alanine interfered with mitosis-associated reduction in MT affinity of XMAP4, and their overexpression affected chromosome movement during anaphase A. These findings indicated that phosphorylation of XMAP4 (probably by p34(cdc2) kinase) is responsible for the decrease in its MT-binding and -stabilizing abilities during mitosis, which are important for chromosome movement during anaphase A.     

Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function.

Eukaryotic cell cycle progression requires the periodic activation and inactivation of a protein-serine/threonine kinase which in fission yeast is encoded by the cdc2+ gene. The activity of this gene product, p34cdc2, is controlled by numerous interactions with other proteins and by its phosphorylation state. In fission yeast, p34cdc2 is phosphorylated on two sites, one of which has been identified as Tyr15. Dephosphorylation of Tyr15 regulates the initiation of mitosis. To understand more completely the regulation of p34cdc2 kinase activity, we have identified the second site of phosphorylation as Thr167, a residue conserved amongst all p34cdc2 homologues. By analysing the phenotypes of cells expressing various position 167 mutations and performing in vitro experiments, we establish that Thr167 phosphorylation is required for p34cdc2 kinase activity at mitosis and is involved in the association of p34cdc2 with cyclin B. Dephosphorylation of Thr167 might also play a role in the exit from mitosis.     

Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition.

Progression through the cell cycle is monitored at two major points: during the G1/S and the G2/M transitions. In most cells, the G2/M transition is regulated by the timing of p34cdc2 dephosphorylation which results in the activation of the kinase activity of the cdc2-cyclin B complex. The timing of p34cdc2 dephosphorylation is determined by the balance between the activity of the kinase that phosphorylates p34cdc2 (wee1 in human cells) and the opposing phosphatase (cdc25C). Both enzymes are regulated and it has been shown that cdc25C is phosphorylated and activated by the cdc2-cyclin B complex. This creates a positive feed-back loop providing a switch used to control the onset of mitosis. Here, we show that another member of the human cdc25 family, cdc25A, undergoes phosphorylation during S phase, resulting in an increase of its phosphatase activity. The phosphorylation of cdc25A is dependent on the activity of the cdc2-cyclin E kinase. Microinjection of anti-cdc25A antibodies into G1 cells blocks entry into S phase. These results indicate that the cdc25A phosphatase is required to enter S phase in human cells and suggest that this enzyme is part of an auto-amplification loop analogous to that described at the G2/M transition. We discuss the nature of the in vivo substrate of the cdc25A phosphatase in S phase and the possible implications for the regulation of S phase entry.     

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.     

Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34^cdc2 phosphorylation site in the consensus S₅₀PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34^cdc2 from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34^cdc2 in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13^suc1-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34^cdc2 kinase. In vivo ³²P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34^cdc2 kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA. Received: 5 September 1996; in revised form: 4 February 1997 / Accepted: 24 February 1997