Characterization of mitotically phosphorylated caldesmon.

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678; Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172). To explore the function of mitosis-specific phosphorylation of caldesmon, in vivo- and in vitro-phosphorylated caldesmons have been characterized. We have found that both in vivo and in vitro phosphorylation of caldesmon causes similar changes in the properties, including reduction in actin, calmodulin, and myosin binding of caldesmon, and a decrease in the inhibition of actomyosin ATPase by caldesmon. Rat non-muscle caldesmon is phosphorylated in vitro up to a ratio of 7 mol/mol of protein. Actin-binding constants of both a high affinity (K a = 1.2 x 10(7) M-1) and a low affinity (K a = 1 x 10(6) M-1) site of unphosphorylated caldesmon are reduced to less than 10(5) M-1 with 5 mol of phosphate incorporation per mol of protein. Actin-bound caldesmon can be phosphorylated by cdc2 kinase, which results in the dissociation of caldesmon from F-actin. Caldesmon has a second myosin-binding site in the C terminus, in addition to the N terminus myosin-binding domain previously reported, because the bacterially expressed C terminus of caldesmon shows binding to myosin. Phosphorylation of the C-terminal fragments decreases their myosin-binding affinity as observed with intact caldesmon. These results suggest that caldesmon loses most of its in vitro functions during mitosis as a result of phosphorylation, which may be required for the reorganization of microfilaments during mitosis.     

Mitosis-specific phosphorylation of caldesmon: possible molecular mechanism of cell rounding during mitosis.

One of the profound changes in cellular morphology during mitosis is a massive alteration in the organization of microfilament cytoskeleton. It has been recently discovered that nonmuscle caldesmon, an actin and calmodulin binding microfilament-associated protein of relative molecular mass Mr = 83,000, is dissociated from microfilaments during mitosis, apparently as a consequence of mitosis-specific phosphorylation. cdc2 kinase, which is a catalytic subunit of MPF (maturation or mitosis promoting factor), is found to be responsible for the mitosis-specific phosphorylation of caldesmon. Because caldesmon is implicated in the regulation of actin myosin interactions and/or microfilament organization, these results suggest that cdc2 kinase directly affects microfilament re-organization during mitosis.     

Phosphorylation of caldesmon by cdc2 kinase

A recent report that mitosis-specific phosphorylation causes the nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678) suggests that this process may contribute to the major structural reorganization of the eukaryotic cell at mitosis. In this study we have demonstrated that smooth muscle caldesmon is phosphorylated in vitro by cdc2 kinase from mitotic phase HeLa cells to 1.2 mol of phosphate/mol of caldesmon. Tryptic maps showed three major phosphorylated spots and approximately equal amounts of phosphorylated Ser and Thr were identified. F-actin or calmodulin in the presence of Ca2+ blocks the phosphorylation of caldesmon. Phosphorylation of caldesmon greatly reduced its binding to F-actin. The phosphorylation sites were located in a 10,000-Da CnBr fragment at the COOH-terminal end of the caldesmon molecule known to house the binding sites for actin and calmodulin (Bartegi A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238). Our finding supports the model that phosphorylation of caldesmon by cdc2 kinase at mitosis may contribute to the disassembly of the microfilament bundles during prophase.     

Caldesmon phosphorylation in actin cytoskeletal remodeling

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.     

Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.     

Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions.

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.     

The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity.     

p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.     

Biochemical Differences between Staurosporine-Induced Apoptosis and Premature Mitosis

Apoptosis is morphologically related to premature mitosis, an aberrant form of mitosis. Staurosporine, a potent protein kinase inhibitor, induces not only apoptotic cell death in a wide variety of mammalian cells but also premature initiation of mitosis in hamster cells that are arrested in S phase by DNA synthesis inhibitors. Here we report on the biochemical differences between the two phenomena commonly caused by staurosporine. Rat 3Y1 fibroblasts that had been arrested in S phase with hydroxyurea underwent apoptosis by treatment with staurosporine, whereas S-phase-arrested CHO cells initiated mitosis prematurely when similarly treated with a low concentration of staurosporine. Chromosome condensation occurred in both apoptosis (3Y1) and premature mitosis (CHO). However, neither formation of mitotic spindles nor mitosis-specific phosphorylation of MPM-2 antigens was observed in apoptosis of 3Y1 cells, unlike premature mitosis of CHO cells. The p34^cdc2kinase activated in normal and prematurely mitotic cells remained inactive in the apoptotic cells, probably because the active cyclin B/p34^cdc2complex was almost absent in the S-phase-arrested 3Y1 cells. The absence of intracellular activation of p34^cdc2in apoptosis was confirmed by immunohistochemical analyses using a specific antibody raised against Ser⁵⁵-phosphorylated vimentin which is specifically phosphorylated by p34^cdc2during M phase. Furthermore, phosphorylation of histones H1 and H3, which is associated with mitotic chromosome condensation, did not occur in the apoptotic cells. These results indicate that the two phenomena, staurosporine-induced apoptosis and premature mitosis, are different in their requirement for p34^cdc2kinase activation and histone phosphorylation.     

Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis.

We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo.     

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.     

Localization of caldesmon and its dephosphorylation during cell division

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments during prometaphase. (Yamashiro, S., Y. Yamakita, R. Ishikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678; Yamashiro, S., Y. Yamakita, H. Hosoya, and F. Matsumura. 1991. Nature (Lond.) 349:169-172). To explore the functions of caldesmon phosphorylation during cytokinesis, we have examined the relationship between the phosphorylation level, actin- binding, and in vivo localization of caldesmon in cultured cells after their release of metaphase arrest. Immunofluorescence studies have revealed that caldesmon is localized diffusely throughout cytoplasm in metaphase. During early stages of cytokinesis, caldesmon is still diffusely present and not concentrated in contractile rings, in contrast to the accumulation of actin in cleavage furrows during cytokinesis. In later stages of cytokinesis, most caldesmon is observed to be yet diffusely localized although some concentration of caldesmon is observed in cortexes as well as in cleavage furrows. When daughter cells begin to spread, caldesmon shows complete colocalization with F- actin-containing structures. These observations are consistent with changes in the levels of microfilament-associated caldesmon during synchronized cell division. Caldesmon is missing from microfilaments in prometaphase cells arrested by nocodazole treatment, as shown previously (Yamashiro, S., Y. Yamakita, R. Iskikawa, and F. Matsumura. 1990. Nature (Lond.). 344:675-678). The level of microfilament- associated caldesmon stays low (12% of that of interphase cells) when some cells start cytokinesis at 40 min after the release of metaphase arrest. When 60% of cells finish cytokinesis at 60 min, the level of microfilament-associated caldesmon is recovered to 50% of that of interphase cells. The level of microfilament-associated caldesmon is then gradually increased to 80% when cells show spreading at 120 min. Dephosphorylation appears to occur during cytokinesis. It starts when cells begin to show cytokinesis at 40 min and completes when most cells finish cytokinesis at 60 min. These results suggest that caldesmon is not associated with microfilaments of cleavage furrows at least in initial stages of cytokinesis and that dephosphorylation of caldesmon appears to couple with its reassociation with microfilaments. Because caldesmon is known to inhibit actomyosin ATPase and/or regulate actin assembly, its continued dissociation from microfilaments may be required for the assembly and/or activation of contractile rings.     

Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).     

Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.

The cdc25 phosphatase is a mitotic inducer that activates p34cdc2 at the G2/M transition by dephosphorylation of Tyr15 in p34cdc2. cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34cdc2 and cyclin B/p34cdc2 at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34cdc2 kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34cdc2. These mutations inhibit the activation of cdc25 by phosphorylation with p34cdc2 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34cdc2 in cdc25-immunodepleted oocyte extracts, suggesting that a positive feed-back loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34cdc2 that induces abrupt entry into mitosis in vivo.     

Phosphorylation of dis2 protein phosphatase at the C-terminal cdc2 consensus and its potential role in cell cycle regulation.

We show that the fission yeast dis2 protein phosphatase, which is highly similar to mammalian type 1 phosphatase, is a phosphoprotein containing phosphoserine (phospho-S) and threonine (phospho-T). It has several phosphorylation sites, two of which locate in the C-terminus. Phospho-T was abolished in the alanine substitution mutant at the C-terminal T316, which is conserved as a residue in the cdc2 consensus, TPPR, in a number of type 1-like phosphatases. In G2-arrested cdc2-L7 cells, the degree of T316 phosphorylation was reduced, whereas it was enhanced in metaphase-arrested nuc2-663 mutant cells. Phospho-T was produced in dis2 by fission yeast cdc2 kinase, but not in the substitution mutant A316, indicating that the T316 residue was the site for cdc2 kinase in vitro. Phosphatase activity of wild type dis2 was reduced by incubation with cdc2 kinase, but that of mutant dis2-A316 was not. Phosphorylation of T316 hence has a potential significance in cell cycle control in conjunction with cdc2 kinase activation and inactivation. Overexpression phenotypes of wild type dis2+, sds21+ and mutant dis2-A316, sds21-TPPR genes were consistent with negative regulation of dis2 by phosphorylation. This type of regulation would explain why cells harboring the dis2-11 mutation enter mitosis but fail to exit from it.     

Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts

We have examined the effects of elevating the intracellular levels of p34cdc2 kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of p34cdc2 kinase or p34cdc2 kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, p34cdc2 kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that p34cdc2 kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis.     

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.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans.

Previous work has shown that nimA encodes a cell cycle regulated protein kinase that is required along with the p34cdc2 histone H1 kinase (MPF) for mitosis in Aspergillus nidulans. We have now identified two other gene products required for mitosis in A.nidulans. nimT encodes a protein similar to the fission yeast cdc25 tyrosine phosphatase and is required for the conversion of pre-MPF to MPF and nimE encodes a B-type cyclin which is a subunit of MPF. A new genetic interaction between nimEcyclinB and nimTcdc25 type genes is reported. Increased copy number of nimEcyclinB can suppress mutation of nimTcdc25 and also lead to increased accumulation of tyrosine phosphorylated p34cdc2 (pre-MPF). This biochemical observation suggests an explanation for the genetic complementation. If nimEcyclinB recruits p34cdc2 for tyrosine phosphorylation to form pre-MPF it follows that increased expression of nimEcyclinB would increase the level of pre-MPF. The increased level of pre-MPF generated may then allow the mutant nimTcdc25 protein to convert enough pre-MPF to MPF and thus permit some mitotic progression. We also demonstrate that correct cell cycle regulation by the p34cdc2 protein kinase pathway is essential for correct developmental progression in A.nidulans.