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.     

In vivo phosphorylation of the lamin B receptor. Binding of lamin B to its nuclear membrane receptor is affected by phosphorylation

Previous studies have shown that the nuclear envelope of avian erythrocytes contains a 58-kDa integral membrane protein (p58) which serves as a receptor for the karyoskeletal protein lamin B (Worman, J. H., Yuan, J., Blobel, G., and Georgatos, S. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8531-8534). We now demonstrate that p58 is phosphorylated in vivo at serine residues and that its phosphorylation is stimulated by isoproterenol in a dose-dependent fashion. We further show that dephosphorylation of p58 reduces significantly its binding to lamin B. These data suggest that phosphorylation may constitute one of the major mechanisms regulating the lamina-nuclear membrane interactions.     

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.     

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.     

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.     

Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase

As cells enter mitosis, the protein-tyrosine kinase, p60c-src, is known to be extensively phosphorylated on threonine in its amino-terminal region. In the present work, extracts of mitotic cells were searched for the protein kinase responsible for this phosphorylation. HeLa cells and Xenopus eggs were found to contain a mitosis-specific protein kinase activity capable of phosphorylating highly purified p60c-src in vitro on threonine residues. Tryptic phosphopeptide maps indicate that the mitotic HeLa kinase phosphorylates the same sites in vitro as those used during mitosis in vivo. In addition, this mitotic HeLa kinase comigrates on gel filtration with p34cdc2-associated histone H1 kinase, a well known regulator of mitotic events. Finally, antibodies to the C-terminal peptide of human p34cdc2 specifically deplete p60c-src-phosphorylating activity from mitotic extracts. These results suggest that p60c-src may act as an effector of p34cdc2 in certain mitotic processes.     

The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane

The lamin B receptor (LBR) is a polytopic integral membrane protein localized exclusively in the inner nuclear membrane domain of the nuclear envelope. Its cDNA deduced primary structure consists of a highly charged amino-terminal domain of 205 residues that faces the nucleoplasm followed by a hydrophobic domain with eight potential transmembrane segments. To identify determinants that sort LBR from its site of integration (RER and outer nuclear membrane) to the inner nuclear membrane, we prepared full-length, truncated, and chimeric cDNA constructs of chick LBR, transfected these into mammalian cells and detected the expressed protein by immunofluorescence microscopy using appropriate antibodies. Surprisingly, we found that the determinants for sorting of LBR to the inner nuclear membrane reside in a region comprising its first transmembrane sequence plus flanking residues on either side. The other transmembrane regions as well as the nucleoplasmic domain are not required for sorting. We propose that the first transmembrane segment of LBR interacts specifically with another transmembrane segment and consider several mechanisms by which such specific interaction could result in sorting to the inner nuclear membrane.     

p34cdc2 kinase-mediated release of lamins from nuclear ghosts is inhibited by cAMP-dependent protein kinase.

During mitosis the lamins are found in a hyperphosphorylated and soluble state. p34cdc2 kinase (MPF), a protein kinase complex with a pivotal role during mitosis, has been found to phosphorylate the lamins and, in some cases, though not all, to cause depolymerization of the lamina in vitro. Due to the variety of protein interactions in the lamina, there is a probable requirement for multiple enzyme activities to effect its breakdown in mitosis. Using nuclear ghosts as substrate, we have fractionated a Xenopus mitotic extract into a lamin-releasing fraction (p34cdc2 kinase) and a fraction that inhibits p34cdc2 kinase-mediated lamin release if the nuclear ghosts are first preincubated in it. The lamin-release-inhibiting activity in the p34cdc2 kinase-depleted mitotic extract is, in turn, inhibited if PKI, a protein kinase inhibitor specific for PKA, is included in the preincubation reaction mixture. Furthermore, a similar degree of inhibition can be achieved by using purified PKA to preincubate the nuclear ghosts. This suggests that dephosphorylation of PKA substrate sites is necessary for lamin depolymerization.     

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.     

The Arabidopsis functional homolog of the p34cdc2 protein kinase.

The p34cdc2 protein kinase is a key component of the eukaryotic cell cycle, which is required for G1 to S-phase transition and for entry into mitosis. Using a 380-base pair DNA fragment obtained by polymerase chain reaction amplification from an Arabidopsis thaliana flower cDNA library as a probe, we isolated and sequenced a cdc2-homologous cDNA from Arabidopsis. The encoded polypeptide has extensive homology with cdc2-like kinases. Furthermore, when expressed in a CDC28ts Saccharomyces strain, it partially restores the capacity to grow at 36 degrees C, indicating that the plant cDNA is a functional homolog of the p34cdc2 kinase. Genomic hybridization demonstrated that there is one copy of the cdc2 gene per Arabidopsis haploid genome. Using RNA gel blot analysis, we found that cdc2 mRNA is present in all plant organs.     

The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.     

The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope

Using heterochromatin-enriched fractions, we have detected specific binding of mononucleosomes to the N-terminal domain of the inner nuclear membrane protein lamin B receptor. Mass spectrometric analysis reveals that LBR-associated particles contain complex patterns of methylated/acetylated histones and are devoid of "euchromatic" epigenetic marks. LBR binds heterochromatin as a higher oligomer and forms distinct nuclear envelope microdomains in vivo. The organization of these membrane assemblies is affected significantly in heterozygous ic (ichthyosis) mutants, resulting in a variety of structural abnormalities and nuclear defects.     

Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis

During mammalian spermiogenesis, histones are replaced by transition proteins, which are in turn replaced by protamines P1 and P2. P1 protamine contains a short arginine/serine-rich (RS) domain that is highly phosphorylated before being deposited into sperm chromatin and almost completely dephosphorylated during sperm maturation. We now demonstrate that, in elongating spermatids, this phosphorylation is required for the temporal association of P1 protamine with lamin B receptor (LBR), an inner nuclear membrane protein that also possesses a stretch of RS dipeptides at its nucleoplasmic NH(2)-terminal domain. Previous studies have shown that the cellular protein p32 also binds tightly to the unmodified RS domain of LBR. Extending those findings, we now present evidence that p32 prevents phosphorylation of LBR and furthermore that dissociation of this protein precedes P1 protamine association. Our data suggest that docking of protamine 1 to the nuclear envelope is an important intermediate step in spermiogenesis and reveal a novel role for SR protein kinases and p32.     

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.     

Staurosporine is a potent inhibitor of p34cdc2 and p34cdc2-like kinases.

We previously demonstrated that nontransformed cells arrest in the G1 phase of the cell cycle when treated with low concentrations (21 nM) of staurosporine (1). Both normal and transformed cells are blocked in the G2 phase of the cell cycle when treated with higher concentrations (160 nM) of staurosporine (1,2). In the present study, we show that staurosporine inhibits the activity of fractionated p34cdc2 and p34cdc2-like kinases with IC50 values of 4-5 nM. We propose that the G2 phase arrest in the cell cycle caused by staurosporine is due, at least in part, to the inhibition of the p34cdc2 kinases.     

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.     

Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.