p34cdc2 acts as a lamin kinase in fission yeast

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.     

Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase.

The hexameric ATPase p97/yeast Cdc48p has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle pole body function, and ubiquitin-dependent protein degradation. p97/Cdc48p contains two conserved consensus p34cdc2 kinase phosphorylation sites within its second ATP binding domain. This domain is likely to play a role in stabilising the hexameric form of the protein. We therefore investigated whether p97 could be phosphorylated by p34cdc2 kinase in vitro, and whether phosphorylation might influence the oligomeric status of p97. Monomeric, but not hexameric, p97 was phosphorylated by p34cdc2 kinase, as was the p97-associated protein p47. However, phosphorylation by p34cdc2 kinase did not impair subsequent re-hexamerisation of p97, implying that the phosphorylated residue(s) are not critical for interaction between p97 monomers. Moreover, p97 within both interphase and mitotic cytosols was almost exclusively hexameric, suggesting that the activity of p97 is not regulated during mitosis by influencing the extent of oligomerisation.     

Subcellular Distribution and Phosphorylation of the Nuclear Localization Signal Binding Protein, NBP60

We previously purified a nuclear localization signal binding protein, NBP60, from rat liver (1993,J. Biochem.113, 308–313). In this study, the subcellular localization of NBP60 was examined using anti-NBP60. Most NBP60 was found to be localized in the nuclear envelope fraction of rat liver obtained on cell fractionation followed by immunoblotting. Staining of the nuclei of cultured cells by the antibody was observed on immunofluorescence microscopy. NBP60 was widely detected in rat nuclear fractions prepared from other tissues and also in nuclei of cultured cells derived from other species. It was shown by immunoelectron microscopy that most NBP60 is present in the nuclear envelope and at least some of that is present on nuclear pore complexes. Although NBP60 was localized in the nuclear envelope in interphase cells, it diffused into the cytoplasm in the mitotic phase. The purified NBP60 was highly phosphorylated by a cdc2 mitotic kinase, whereas nuclear pore proteins p144, p62, p60, and p54 were not phosphorylated by the kinase directly. NBP60 was also phosphorylated by protein kinase A, calmodulin-dependent protein kinase II, and casein kinase II. The phosphorylation of NBP60 by cdc2 kinase and/or the other kinases may be related to the change in the protein's location during the mitotic phase.     

A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.

While the p34cdc2 kinase is considered to be a critical regulator of mitosis, its function has not yet been directly linked to one of the key events during the onset of mitosis: nuclear envelope breakdown. Here we show that a major structural protein of the nuclear envelope, lamin B2, is phosphorylated by p34cdc2. Results from two-dimensional phosphopeptide mapping experiments demonstrate that the p34cdc2-specific phosphopeptides represent both mitotic and interphase specific phosphorylations of lamin B2 and include the major interphase phosphorylation site. In mitotic cells we detected two distinct forms of lamin B2 which differ in electrophoretic mobility and in degree of phosphorylation. The phosphorylation pattern of lamin B2 generated in vitro by p34cdc2 was more closely related to the less phosphorylated mitotic lamin B2, suggesting that another kinase(s) in addition to p34cdc2 is involved in generating the mitotic phosphorylation pattern. In addition, we show that treatment of interphase cells with okadaic acid, a potent phosphatase inhibitor, leads to the acquisition of mitosis-specific phosphopeptides and can reversibly increase the detergent-solubility of lamin B2. However, the M-phase-like phosphorylation of lamin B2 in itself is not sufficient to induce its disassembly from the nuclear lamina suggesting that an additional event(s) besides phosphorylation is required.     

Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.     

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.     

A deficiency in the mechanism for p34cdc2 protein kinase activation in mouse embryos arrested at 2-cell stage.

Mouse embryos of the ddY strain fertilized in vitro undergo the first cleavage to the 2-cell stage but not the second cleavage even 45 hr after insemination (2-cell block). We examined the phosphorylation state of p34cdc2 and histone H1 kinase activity in mouse 2-cell embryos to investigate the relationship of p34cdc2 with 2-cell block. In the first mitotic cell cycle, the amount of phosphorylated forms of p34cdc2, which were detected as the bands of retarded mobility on SDS-PAGE followed by immunoblotting with anti-p34cdc2 antibody, increased during interphase and abruptly decreased at M phase. Concomitant with this dephosphorylation, histone H1 kinase activity was increased. After the embryos cleaved to the 2-cell stage, the amounts of phosphorylated forms of p34cdc2 increased up to 33 hr after insemination. However, the activation of histone H1 kinase did not occur and the states of phosphorylation of p34cdc2 did not show any significant changes until 45 hr. In contrast, 2-cell embryos of B6C3F1 mice, which do not show a 2-cell block and develop normally to blastocysts in vitro, exhibit the dephosphorylation of p34cdc2 and an increase in histone H1 kinase activity between 31 and 45 hr after insemination. When the ddY mouse embryos arrested at the 2-cell stage were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, the dephosphorylation of p34cdc2 occurred and histone H1 kinase activity increased. The chromosomes of these embryos stained with 4',6'-diamidino-2-phenylindole revealed the initiation of condensation. These results suggest that 2-cell-blocked embryos contain enough p34cdc2 to induce mitotic events but the protein remains in a latent form.     

Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues.

Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo. The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state. At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus. We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner. A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage. The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components. Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention. Both wild-type xnf7 and xnf7thr-glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention. We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage.     

p34cdc2 is physically associated with and phosphorylated by a cdc2-specific tyrosine kinase.

The mammalian homologue of the yeast cdc2 gene encodes a 34-kilodalton serine/threonine kinase that is a subunit of M phase-promoting factor. Recent studies have shown that p34cdc2 is also a major tyrosine-phosphorylated protein in HeLa cells and that its phosphotyrosine content is cell cycle regulated and related to its kinase activity. Here, we show that cdc2 is physically associated with and phosphorylated in vitro by a highly specific tyrosine kinase. Tyrosine phosphorylation of cdc2 in vitro occurs at tyrosine 15, the same site that is phosphorylated in vivo. The association between the two kinases takes place in the cytosolic compartment and involves cyclin B-associated cdc2. Evidence is presented that a substantial fraction of cytosolic cdc2 is hypophosphorylated, whereas nuclear cdc2 is hyperphosphorylated. Finally, we show that the tyrosine kinase associated with cdc2 may be a 67-kilodalton protein and is distinct from src, abl, fms, and other previously reported tyrosine kinases.     

M-phase-specific phosphorylation and structural rearrangement of the cytoplasmic cross-linking protein plectin involve p34cdc2 kinase.

Plectin, a widespread and abundant cytoskeletal cross-linking protein, serves as a target for protein kinases throughout the cell cycle, without any significant variation in overall phosphorylation level. One of the various phosphorylation sites of the molecule was found to be phosphorylated preferentially during mitosis. By in vivo phosphorylation of ectopically expressed plectin domains in stably transfected Chinese hamster ovary cells, this site was mapped to the C-terminal repeat 6 domain of the polypeptide. The same site has been identified as an in vitro target for p34cdc2 kinase. Mitosis-specific phosphorylation of plectin was accompanied by a rearrangement of plectin structures, changing from a filamentous, largely vimentin-associated state in interphase to a diffuse vimentin-independent distribution in mitosis as visualized by immunofluorescence microscopy. Subcellular fractionation studies showed that in interphase cells up to 80% of cellular plectin was found associated with an insoluble cell fraction mostly consisting of intermediate filaments, while during mitosis the majority of plectin (> 75%) became soluble. Furthermore, phosphorylation of purified plectin by p34cdc2 kinase decreased plectin's ability to interact with preassembled vimentin filaments in vitro. Together, our data suggest that a mitosis-specific phosphorylation involving p34cdc2 kinase regulates plectin's cross-linking activities and association with intermediate filaments during the cell cycle.     

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.     

In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. Phosphorylation of lamin proteins is believed to cause lamina disassembly during meiotic and mitotic M phase, but the M phase-specific lamin kinase has not been identified. Here we show that the cdc2 kinase, a major element implicated in controlling the eukaryotic cell cycle, phosphorylates chicken B-type lamins in vitro on sites that are specifically phosphorylated during M phase in vivo. Concomitantly, cdc2 kinase is capable of inducing lamina depolymerization upon incubation with isolated nuclei. One of the target sites of cdc2 kinase is identified as a motif (SPTR) conserved in the N-terminal domain of all lamin proteins. These results lead us to propose that mitotic disassembly of the nuclear lamina results from direct phosphorylation of lamins by cdc2 kinase.     

Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C gamma and neurogranin/RC3 genes

A 20-kDa DNA-binding protein that binds the AT-rich sequences within the promoters of the brain-specific protein kinase C (PKC) gamma and neurogranin/RC3 genes has been characterized as chromosomal nonhistone high-mobility-group protein (HMG)-I. This protein is a substrate of PKC alpha, beta, gamma, and delta but is poorly phosphorylated by PKC epsilon and zeta. Two major (Ser44 and Ser64) and four minor phosphorylation sites have been identified. The extents of phosphorylation of Ser44 and Ser64 were 1:1, whereas those of the four minor sites all together were <30% of the major one. These PKC phosphorylation sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr53 and Thr78. Phosphorylation of HMG-I by PKC resulted in a reduction of DNA-binding affinity by 28-fold as compared with 12-fold caused by the phosphorylation with cdc2 kinase. HMG-I could be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a >100-fold reduction in binding affinity. The two cdc2 kinase phosphorylation sites of HMG-I are adjacent to the N terminus of two of the three predicted DNA-binding domains. In comparison, one of the major PKC phosphorylation sites, Ser64, is adjacent to the C terminus of the second DNA-binding domain, whereas Ser44 is located within the spanning region between the first and second DNA-binding domains. The current results suggest that phosphorylation of the mammalian HMG-I by PKC alone or in combination with cdc2 kinase provides an effective mechanism for the regulation of HMG-I function.     

The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.     

Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates.

The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis.     

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.