Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.     

APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit

Cyclin degradation is central to regulation of the cell cycle. Mitotic exit was proposed to require degradation of the S phase cyclin Clb5 by the anaphase-promoting complex activated by Cdc20 (APC(Cdc20)). Furthermore, Clb5 degradation was thought to be necessary for effective dephosphorylation and activation of the APC regulatory subunit Cdh1 (also known as Hct1) and the cyclin-dependent kinase inhibitor Sic1 by the phosphatase Cdc14, allowing mitotic kinase inactivation and mitotic exit. Here we show, however, that spindle disassembly and cell division occur without significant APC(Cdc20)-mediated Clb5 degradation, as well as in the absence of both Cdh1 and Sic1. We find instead that destruction-box-dependent degradation of the mitotic cyclin Clb2 is essential for mitotic exit. APC(Cdc20) may be required for an essential early phase of Clb2 degradation, and this phase may be sufficient for most aspects of mitotic exit. Cdh1 and Sic1 may be required for further inactivation of Clb2-Cdk1, regulating cell size and the length of G1.     

Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast

The coordination of somatic cell division with cell size must be accomplished by the accumulation of mitotic inducers or the dilution, in the course of cell growth, of mitotic inhibitors. In fission yeast (Schizosaccharomyces pombe), cell size at mitosis is determined by expression of the cdc25+ and nim1+ inducer genes and of the inhibitor gene wee1+, which between them regulate the M-phase protein kinase p34cdc2. We now report that both the phosphoprotein product of cdc25+ (p80cdc25, with apparent relative molecular mass 80,000) and the corresponding messenger RNA increase in concentration as cells proceed through interphase, peaking at mitosis. We propose that the cell-cycle timing of mitosis in somatic cells is regulated by the cyclic accumulation of the cdc25 mitotic inducer, which on reaching a critical level results in activation of p34cdc2 protein kinase. Accumulation of this inducer could play a part in coordinating cell division with growth.     

In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase.

The protein kinase MAP kinase, also called MAP2 kinase, is a serine/threonine kinase whose activation and phosphorylation are induced by a variety of mitogens, and which is thought to have a critical role in a network of protein kinases in mitogenic signal transduction. A burst in kinase activation and protein phosphorylation may also be important in triggering the dramatic reorganization of the cell during the transition from interphase to mitosis. The interphase-metaphase transition of microtubule arrays is under the control of p34cdc2 kinase, a central control element in the G2-M transition of the cell cycle. Here we show that a Xenopus kinase, closely related to the mitogen-activated mammalian MAP kinase, is phosphorylated and activated during M phase of meiotic and mitotic cell cycles, and that the interphase-metaphase transition of microtubule arrays can be induced by the addition of purified Xenopus M phase-activated MAP kinase or mammalian mitogen-activated MAP kinase to interphase extracts in vitro.     

Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src.

The tyrosine kinasee activity of p60c-src, the protein product of the c-src gene, increases during mitosis; this may be important in initiating at least some of the cellular changes that occur during this phase of the cell cycle. Although there is evidence that p60c-src is phosphorylated at several sites during mitosis, phosphorylation in vitro does not increase its kinase activity. We now report that the kinase activity of a p60c-src mutant with residue tyrosine 527 changed to phenylanine does not change during the cell cycle, suggesting that changes in the phosphorylation state of this residue may be responsible for the activation of p60c-src at mitosis. Although changes in phosphorylation at Tyr 527 cannot be detected with the wild-type protein we find that phosphorylation at Tyr 527 of a mutant with reduced kinase activity decreases threefold during mitosis. On the basis of these results we suggest that activation of p60c-src at mitosis results from decreased phosphorylation on Tyr 527, and that p60c-src may be or may activate the kinase that phosphorylates Tyr 527.     

Activation at M-phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2+

In both starfish and amphibian oocytes, the activity of a major protein kinase which is independent of Ca2+ and cyclic nucleotides increases dramatically at meiotic and mitotic nuclear divisions. The in vivo substrates of this kinase are unknown, but phosphorylation of H1 histone can be used as an in vitro assay. We have purified this kinase from starfish oocytes. The major band in the most highly purified preparation contained a polypeptide of relative molecular mass (Mr) 34,000 (34K). This is the same size as the protein kinase encoded by cdc2+, which regulates entry into mitosis in fission yeast and is a component of MPF purified from Xenopus. Here, we show that antibodies against p34 recognize the starfish 34K protein and propose that entry into meiotic and mitotic nuclear divisions involves activation of the protein kinase encoded by a homologue of cdc2+. Given the wide occurrence of cdc2+ homologues from budding yeast to Xenopus and human cells, this activation may act as a common mechanism controlling entry into mitosis in eukaryotic cells.     

Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates

Cell-cycle events are controlled by cyclin-dependent kinases (CDKs), whose periodic activation is driven by cyclins. Different cyclins promote distinct cell-cycle events, but the molecular basis for these differences remains unclear. Here we compare the specificity of two budding yeast cyclins, the S-phase cyclin Clb5 and the M-phase cyclin Clb2, in the phosphorylation of 150 Cdk1 (Cdc28) substrates. About 24% of these proteins were phosphorylated more efficiently by Clb5-Cdk1 than Clb2-Cdk1. The Clb5-specific targets include several proteins (Sld2, Cdc6, Orc6, Mcm3 and Cdh1) involved in early S-phase events. Clb5 specificity depended on an interaction between a hydrophobic patch in Clb5 and a short sequence in the substrate (the RXL or Cy motif). Phosphorylation of Clb5-specific targets during S phase was reduced by replacing Clb5 with Clb2 or by mutating the substrate RXL motif, confirming the importance of Clb5 specificity in vivo. Although we did not identify any highly Clb2-specific substrates, we found that Clb2-Cdk1 possessed higher intrinsic kinase activity than Clb5-Cdk1, enabling efficient phosphorylation of a broad range of mitotic Cdk1 targets. Thus, Clb5 and Clb2 use distinct mechanisms to enhance the phosphorylation of S-phase and M-phase substrates.     

Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis

One of the profound changes in cellular morphology which occurs during mitosis is a massive alteration in the organization of the microfilament cytoskeleton. This change, together with other mitotic events including nuclear membrane breakdown, chromosome condensation and formation of mitotic spindles, is induced by a molecular complex called maturation promoting factor. This consists of at least two subunits, a polypeptide of relative molecular mass 45,000-62,000 (Mr 45-62K) known as cyclin, and a 34K catalytic subunit which has serine/threonine kinase activity and is known as cdc2 kinase. Non-muscle caldesmon, an 83K actin- and calmodulin-binding protein, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. We now report that cdc2 kinase phosphorylates caldesmon in vitro principally at the same sites as those phosphorylated in vivo during mitosis, and that phosphorylation reduces the binding affinity of caldesmon for both actin and calmodulin. Because caldesmon inhibits actomyosin ATPase, our results suggest that cdc2 kinase directly causes microfilament reorganization during mitosis.     

Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle

Genetic studies in the fission yeast Schizosaccharomyces pombe have established that a critical element required for the G2----M-phase transition in the cell cycle is encoded by the cdc2+ gene. The product of this gene is a serine/threonine protein kinase, designated p34cdc, that is highly conserved functionally from yeast to man2 and has a relative molecular mass of 34,000 (34 K). Purified maturation-promoting factor (MPF) is a complex of p34cdc2 and a 45K substrate that appears in late G2 phase and is sufficient to drive cells into mitosis. This factor has been identified in all eukaryotic cells, and in vitro histone H1 is the preferred substrate for phosphorylation. The increase in the activity of H1 kinase in M-phase is associated with a large increase in total cell protein phosphorylation which is believed to be a consequence of MPF activation. We show here that the H1 kinase activity of p34cdc2 oscillates during the cell cycle in Xenopus, and maximal activity correlates with the dephosphorylated state of p34cdc2. Direct inactivation of MPF in vitro is accompanied by phosphorylation of p34cdc2 and reduction of its protein kinase activity.     

Ser28磷酸化组蛋白H3在哺乳动物有丝分裂细胞中的动态分布

利用特异性抗Ser28磷酸化组蛋白H3抗体,应用间接免疫荧光标记技术,标记人乳腺癌细胞 (MCF-7)和小鼠成纤维细胞(NIH 3T3),用激光共聚焦显微术研究这两种哺乳动物细胞中Ser28磷酸化组蛋白 H3在有丝分裂过程中的动态分布,以研究Ser28磷酸化组蛋白在细胞有丝分裂过程中的作用.结果表明,Ser28 的磷酸化作用是这两种细胞有丝分裂期的特有事件.组蛋白H3的Ser28磷酸化信号首先出现在早期的核外周, Ser28磷酸化在中期达到高峰,并扩展到染色体的所有部分,后期和末期逐渐减退,随着胞质分裂的完成而消失. 实验结果表明,组蛋白H3 Ser28的磷酸化与有丝分裂染色体的凝集和解凝集过程有着时间和空间上的相关性. Ser28磷酸化使得组蛋白H3氨基末端的正电荷数降低,这可能是导致染色质变构凝集的原因之一.有丝分裂期 间组蛋白H3在Ser28位置磷酸化过程与Ser10相比有明显的差异,因此在动物细胞中,组蛋白H3氨基末端这 两个不同丝氨酸残基的磷酸化可能有不同的生物学功能.     

Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle.

In yeast and somatic cells, mechanisms ensure cell-cycle events are initiated only when preceding events have been completed. In contrast, interruption of specific cell-cycle processes in early embryonic cells of many organisms does not affect the timing of subsequent events, indicating that cell-cycle events are triggered by a free-running cell-cycle oscillator. Here we present evidence for an independent cell-cycle oscillator in the budding yeast Saccharomyces cerevisiae. We observed periodic activation of events normally restricted to the G1 phase of the cell cycle, in cells lacking mitotic cyclin-dependent kinase activities that are essential for cell-cycle progression. As in embryonic cells, G1 events cycled on schedule, in the absence of S phase or mitosis, with a period similar to the cell-cycle time of wild-type cells. Oscillations of similar periodicity were observed in cells responding to mating pheromone in the absence of G1 cyclin (Cln)- and mitotic cyclin (Clb)-associated kinase activity, indicating that the oscillator may function independently of cyclin-dependent kinase dynamics. We also show that Clb-associated kinase activity is essential for ensuring dependencies by preventing the initiation of new G1 events when cell-cycle progression is delayed.     

Calcineurin is required to release Xenopus egg extracts from meiotic M phase

Fertilization induces a transient increase in cytoplasmic Ca2+ concentration in animal eggs that releases them from cell cycle arrest in the second meiotic metaphase. In frog eggs, Ca2+ activates Ca2+/calmodulin-activated kinase, which inactivates cytostatic factor, allowing the anaphase-promoting factor to turn on and ubiquitinate cyclins and securin, which returns the cell cycle to interphase. Here we show that the calcium-activated protein phosphatase calcineurin is also important in this process. Calcineurin is transiently activated after adding Ca2+ to egg extracts, and inhibitors of calcineurin such as cyclosporin A (ref. 8) delay the destruction of cyclins, the global dephosphorylation of M-phase-specific phosphoproteins and the re-formation of a fully functional nuclear envelope. We found that a second wave of phosphatase activity directed at mitotic phosphoproteins appears after the spike of calcineurin activity. This activity disappeared the next time the extract entered M phase and reappeared at the end of mitosis. We surmise that inhibition of this second phosphatase activity is important in allowing cells to enter mitosis, and, conversely, that its activation is required for a timely return to interphase. Calcineurin is required to break the deep cell cycle arrest imposed by the Mos-MAP (mitogen-activated protein) kinase pathway, and we show that Fizzy/Cdc20, a key regulator of the anaphase-promoting factor, is an excellent substrate for this phosphatase.     

The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. We also show that tumour-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and that cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumour suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.