DOI： 10.1006/dbio.1998.8881

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DOI： 10.1006/dbio.1998.8881

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

G2-phase-arrested immature starfish oocytes contain inactive cdc2 kinase and cdc25 phosphatase, and an inactivator for cdc2 kinase. In this system, we have studied how the regulatory balance is tipped toward the initial activation of cdc2 kinase. During the hormone-dependent period (Guerrier, P., and M. Doree. 1975. Dev. Biol. 47:341-348), p34(cdc2) and cdc25 protein are already converted, though not fully, to active forms, whereas the inactivators for cdc2 kinase and cdc25 phosphatase are able to exhibit their activities if the hormone were removed, We produced ''triggered oocytes,'' in which due to a neutralizing anti-cdc25 antibody, the activation of cdc2 kinase is prevented but cdc25 protein is phosphorylated slightly after the maturation-inducing hormonal stimulus. In contrast to control immature oocytes, in triggered oocytes the injected cdc2 kinase is not inactivated, and accordingly the level of cdc2 kinase activity required for meiosis reinitiation is much less. These results imply the presence of a cdc2 kinase activity-independent process(es) that suppresses the inactivator for cdc2 kinase and initially phosphorylates cdc25 protein, although this process is reversible during the initial activation of cdc2 kinase, At the most initial triggering of M-phase, the cdc2 kinase activity-independent process might trip the switch leading to the initial activation of cdc2 kinase. Thereafter, in parallel, the cdc2 kinase-dependent feedback loops described by others may cause further increase in cdc2 kinase activity. We propose that a putative suppressor, which downregulates the inactivator for cdc2 kinase independently of nuclear components, might be a previously unrecognized component of maturation-promoting factor.

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Language：English   Publisher：LIPPINCOTT-RAVEN PUBL  

We identified two major substrates for the proline-directed protein kinases-cdc2 kinase and tau protein kinase II (TPKII)-in the cytosol fraction from rat brains, The molecular masses of the proteins were 80 and 46 kDa, Because the 80-kDa protein was phosphorylated by protein kinase C and was heat stable, we examined the possibility that the protein might be myristoylated alanine-rich C kinase substrate (MARCKS). On the basis of a comparison between the properties of the 80-kDa protein and purified MARCKS, we concluded that the 80-kDa protein is indeed MARCKS, The amounts of phosphate incorporated into MARCKS by protein kinase C, cdc2 kinase, and TPKII were 1.7, 1.4, and 0.6 mol/mol of the protein, respectively. Two-dimensional tryptic peptide mapping indicated that phosphorylation sites by protein kinase C and proline-directed protein kinases completely differed. Only the seryl residue was phosphorylated by protein kinase C, whereas both seryl and threonyl residues were phosphorylated by cdc2 kinase and TPKII. Phosphorylation of MARCKS by protein kinase C inhibited the binding to calmodulin, whereas phosphorylation by cdc2 kinase and TPKII significantly increased the binding to calmodulin. The holoenzyme of protein phosphatase 2A dephosphorylated MARCKS that had been phosphorylated by protein kinase C, cdc2 kinase, or TPKII, whereas calcineurin was unable to dephosphorylate it, These results suggest that cdc2 kinase and TPKII regulate the functions of MARCKS in different ways from protein kinase C.

DOI： 10.1046/j.1471-4159.1995.65020802.x

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Language：English   Publisher：JAPAN BIOCHEMICAL SOC  

The major kinase capable of phosphorylating tan in a porcine brain extract was suggested to be a brain cdc2-like kinase, called cdk5. Tau protein components of microtubules assembled in the brain extract using ATP were phosphorylated to a higher level, and showed a slower electrophoretic mobility than those assembled with GTP. Most of this phosphorylation and electrophoretic mobility shift, that occurred in the brain extract incubated with ATP, were inhibited by butyrolactone I, a specific inhibitor of cdc2 kinase and cdk5. Further, butyrolactone I inhibited phosphorylation of tan exogenously added to the brain extract by approximately 70%. cdk5 purified from porcine brain decreased the electrophoretic mobility of dephosphorylated tau by in vitro phosphorylation of tau to the level present in microtubules polymerized with ATP. cdc2 kinase purified from starfish oocytes also phosphorylated tau and shifted its electrophoretic mobility to an extent greater than that obtained with cdk5. Western blot analysis showed that cdc2 kinase phosphorylated epitopes recognized by SMI31, 33, 34, and tau1 antibodies in tau proteins, while cdk5 phosphorylated the site recognized by SMI33 (corresponding to phosphorylation at Ser235 in the longest human tau isoform) and partially phosphorylated the tau1 site. Phosphorylation experiments performed on tau in brain extracts, in the presence of okadaic acid, suggested the presence of both okadaic acid-sensitive and -insensitive phosphatases acting on phosphorylated Ser235. Rat tau that was prepared immediately after decapitation showed a similar phosphorylation state to tau in microtubules polymerized with ATP, suggesting that tau is relatively phosphorylated in vivo.

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

We previously demonstrated (Ookata et al., 1992, 1993) that the p34(cdc2)/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34(cdc2) kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34(cdc2)/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34(cdc2)/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34(cdc2)/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34(cdc2), cyclin B, and the COOH-terminal domain of MAP4, PA(4), with ATP resulted in intracomplex phosphorylation of PA(4). Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34(cdc2) kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.

DOI： 10.1083/jcb.128.5.849

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DOI： 10.1016/0197-0186(94)00135-H

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DOI： 10.1016/0197-0186(94)00135-H

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DOI： 10.1046/j.1440-169X.1995.t01-1-00011.x

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DOI： 10.1046/j.1440-169X.1995.t01-1-00011.x

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Myogenin, a member of the MyoD family which governs skeletal muscle differentiation, was identified as a pair of phosphorylated bands on SDS-PAGE during myogenesis. The slow migrating form was found to be hyperphosphorylated myogenin. In vitro phosphorylation by CDC2 kinase caused a prominent reduction in electrophoretic mobility of myogenin. Furthermore, we demonstrated that phosphorylation of the serine residue at position 43 contributes to the modification of myogenin in vivo and in vitro resulting in the reduction in electrophoretic mobility. We propose here that a CDC2-like proline-directed kinase regulates myogenin activity through its phosphorylation.

DOI： 10.1016/0014-5793(94)00964-3

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

The microtubular cytoskeleton exhibits a dramatic reorganization, progressing from interphase radial arrays to a mitotic spindle at the G2/M transition. Although this reorganization has been suspected to be caused by maturation promoting factor (MPF: p34cdc2/cyclin B complex), little is known about how p34cdc2 kinase controls microtubule networks. We provide evidence of the direct association of the p34cdc2/cyclin B complex with microtubules in starfish oocytes. Anti-cyclin B staining of detergent-treated oocytes, isolated asters and meiotic spindles revealed fluorescence associated with microtubule fibers, chromosomes and centrosomes. Microtubules prepared from starfish oocytes were associated with cyclin B and p34cdc2 proteins. Microtubule-bound p34cdc2 and cyclin B were released from microtubules by a high-salt solution and possessed a complex form as shown by the adsorption to suc1-beads and by immunoprecipitation with the anti-cyclin B antibody. The p34cdc2/cyclin B complex associated to microtubules had high histone H1 kinase activity at meiotic metaphase. However, it was not necessary for the p34cdc2/cyclin B complex to be active for microtubule binding, as an inactive form in immature oocytes was also observed to bind to microtubules. The coprecipitation of suc1-column purified p34cdc2/cyclin B with purified porcine brain microtubules in the presence of starfish oocyte microtubule-associated proteins (MAPs) indicates that the association of p34cdc2/cyclin B with microtubules in vitro is mediated by MAPs.

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Tau protein kinase II purified from a bovine brain tau protein fraction (Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T., and Imahori, K. (1992) J. Biol. Chem. 267, 10897-10901) was shown to have a similar substrate specificity to cdc2 kinase in that both phosphorylate neurofilament (NF) proteins. Tau protein kinase II recognized the dephosphorylated form of the heavy subunit of NF (NF-H) as a predominant substrate. The substrate was phosphorylated to the same extent with tau protein kinase II as with cdc2 kinase. Upon phosphorylation, the electrophoretic mobility of the NF-H on SDS-polyacrylamide gel electrophoresis changed to the position of the phosphorylated form. A synthetic peptide containing a KSPXK sequence was by far a better substrate for tau protein kinase II than that containing a KSPXX sequence, as was also observed with cdc2 kinase. NF-H lost its microtubule-associating ability upon phosphorylation with tau protein kinase II as well as with cdc2 kinase. Although anti-PSTAIR antibody (PSTAIR is an amino acid sequence commonly found in cdc2 and several cdc2-related kinases) failed to react with tau protein kinase II, tau protein kinase II bound to p13suc1-Sepharose beads (p13suc1 is a yeast protein known to bind to cdc2 kinase).

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

We have studied the regulation of microtubule nucleating activity of the centrosome using cell-free extracts from Xenopus eggs. We found that the number of microtubules per centrosome increases dramatically with time during incubation of isolated centrosomes in interphasic egg extracts prepared 20-30 minutes after electric activation of cytostatic factor (CSF)-arrested eggs. The increase in microtubule nucleation was still conspicuous even when KCl-treated centrosomes (centrosomes stripped of their microtubule nucleating activity by 1 M KCl treatment) were incubated in interphasic extracts. Electron microscopy and immunostaining by anti-gamma-tubulin and 5051 human anti-centrosome antibodies revealed that pericentriolar material (PCM) was accumulated during the increase in microtubule nucleation from centrosomes in interphasic extracts, suggesting regulation of centrosomal activity by PCM accumulation. The ability of egg extracts to activate microtubule nucleation from centrosomes was also assumed to be regulated by phosphorylation, since addition of protein kinase inhibitors into interphasic extracts totally blocked the increase in microtubule nucleation from the KCl-treated centrosome. The ability of CSF-arrested mitotic extracts to increase microtubule nucleation from KCl-treated centrosomes was 3.5- to 5-fold higher than that of interphasic extracts, while PCM accumulation in mitotic extracts seemed to be similar to that in interphasic extracts. The increase in microtubule nucleation from KCl-treated centrosomes was strikingly enhanced by the addition of purified p34cdc2/cyclin B complex to interphasic extracts, but not by MAP kinase, which is activated downstream of p34cdc2/cyclinB. These results suggest two pathways activating centrosomal activity in egg extracts: accumulation of PCM and phosphorylation mediated by p34cdc2/cyclin B.

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DOI： 10.1006/bbrc.1993.2121

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DOI： 10.1006/bbrc.1993.2121

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DOI： 10.1006/bbrc.1993.1138

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DOI： 10.1006/bbrc.1993.1138

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 m NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca2+-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the, dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides.

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Language：English   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated post-translationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.

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