Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1

Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. Here we propose that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. We show by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. We conclude that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA.     

The Cdc14 phosphatase is functionally associated with the Dbf2 protein kinase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Cdc14 protein phosphatase and Dbf2 protein kinase have been implicated to act during late M phase, but their functions are not known. We report here that CDC14 is a low-copy suppressor of the dbf2-2 mutation at 37 degrees C. The kinase activity of Dbf2 accumulated at a high level, in vivo, during a cdc14 arrest and was also much higher in cdc14 mutant cells at the permissive temperature of growth, therefore in cycling mutant cells than in cycling wild-type cells. This correlated with the accumulation of the more slowly migrating form of Dbf2, previously shown to correspond to the hyperphosphorylated form of the protein. The finding that the dbf2-2 mutation could be rescued following overproduction of catalytically inactive forms of Cdc14 suggested that the control of Dbf2 activity by Cdc14 might be only indirect and independent of Cdc14 phosphatase activity. However, it was found that Cdc14 could form oligomers within the cell, thus leaving open the possibility that catalytically inactive Cdc14 might associate with wild-type Cdc14 and rescue dbf2-2 in a phosphatase-dependent manner. We confirmed that overexpression of CDC14 could rescue mutations in CDC15, which encodes another kinase also implicated to act in late M phase. Cells of a cdc15-2 dbf2-2 double mutant died at temperatures much lower than did either single mutant, whereas there was only a slight additive phenotype in the cdc14-1 dbf2-2 and cdc14-1 cdc15-2 double mutant cells. Finally, functional association between Cdc14 and Dbf2 (and also Cdc15) was confirmed by the finding that the cdc14, dbf2 and cdc15 mutations could be partially rescued by the addition of 1.2 M sorbitol to the culture medium. Our data are the first to demonstrate a functional link between Cdc14 and Dbf2 based on both biochemical and genetic information.     

Identification of protein phosphatase 1 as a mitotic lamin phosphatase

At the onset of mitosis, the nuclear lamins are hyperphosphorylated leading to nuclear lamina disassembly, a process required for nuclear envelope breakdown and entry into mitosis. Multiple lamin kinases have been identified, including protein kinase C, that mediate mitotic lamin phosphorylation and mitotic nuclear lamina disassembly. Conversely, lamin dephosphorylation is required for nuclear lamina reassembly at the completion of mitosis. However, the protein phosphatase(s) responsible for the removal of mitotic phosphates from the lamins is unknown. In this study, we use human lamin B phosphorylated at mitosis-specific sites as a substrate to identify and characterize a lamin phosphatase activity from mitotic human cells. Several lines of evidence demonstrate that the mitotic lamin phosphatase corresponds to type 1 protein phosphatase (PP1). First, mitotic lamin phosphatase activity is inhibited by high nanomolar concentrations of okadaic acid and the specific PP1 peptide inhibitor, inhibitor-2. Second, mitotic lamin phosphatase activity cofractionates with PP1 after ion exchange chromatography. Third, microcystin-agarose depletes mitotic extracts of both PP1 and lamin phosphatase activity. Our results demonstrate that PP1 is the major mitotic lamin phosphatase responsible for removal of mitotic phosphates from lamin B, a process required for nuclear lamina reassembly.     

DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase

A 35-year-old man affected with pulmonary sarcoidosis had a 12-year history of fatigue and pain in the limbs, with normal neurological examination, except for diffusely absent deep tendon reflexes. Muscle biopsy samples showed multiple noncaseating granulomas, most prominent around the intramuscular nerves, with predominance of CD4+ cells. Intramuscular nerve bundles surrounded by granulomas were immunolabelled with laminin alpha1, alpha2, beta1 and gamma1 chain, and collagen IV. Sural nerve biopsy samples were normal. This patient showed a unique histopathological pattern of sarcoid neuromyopathy characterized by distribution of granulomas or infiltrating cells around intramuscular nerve fibers. The clinical picture, restricted to nonspecific symptoms of fatigue and myalgia, and loss of deep tendon reflexes, correlated well with the selective localization of sarcoid lesions in contiguity with the intramuscular nerves. To our knowledge, this peculiar clinico-pathological correlation has not been reported previously.     

The activity of Cdc14p, an oligomeric dual specificity protein phosphatase from Saccharomyces cerevisiae, is required for cell cycle progression

The essential CDC14 gene of the budding yeast, Saccharomyces cerevisiae, encodes a 62-kDa protein containing a sequence that conforms to the active site motif found in all enzymes of the protein tyrosine phosphatase superfamily. Genetic studies suggest that Cdc14p may be involved in the initiation of DNA replication, but its precise cell cycle function is unknown. Recombinant Cdc14p was produced in bacteria, characterized, and shown to be a dual specificity protein phosphatase. Polyanions such as polyglutamate and double-stranded and single-stranded DNA bind to Cdc14p and affect its activity. Native molecular weights of 131,000 and 169,000 determined by two independent methods indicate that recombinant Cdc14p self-associates in vitro to form active oligomers. The catalytically inactive Cdc14p C283S/R289A mutant is not able to suppress the temperature sensitivity of a cdc14-1(ts) mutant nor replace the wild type gene in vivo, demonstrating that phosphatase activity is required for the cell cycle function of Cdc14p. A distinctive COOH-terminal segment (residues 375-551) is rich in Asn and Ser residues, carries a net positive charge, and contains two tandem 21-residue repeats. This COOH-terminal segment is not required for activity, for oligomerization, or for the critical cell cycle function of Cdc14p.     

Visualization of mitotic radial glial lineage cells in the developing rat brain by Cdc2 kinase-phosphorylated vimentin

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF-alpha on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF-alpha in the presence of interferon-gamma (IFN-gamma) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN-gamma, no increase in Mn-SOD activity was detected. The induction of IFN-gamma-dependent Mn-SOD activity by anti-Fas antibody or TNF-alpha was concentration-dependent; the maximal effect was observed at 1-10 micrograms/ml and 5-10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF-alpha in the presence of IFN-gamma resulted in an additive increase in Mn-SOD activity. Although the addition of 12-o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN-gamma-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF-alpha. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN-gamma and that TPA augments the process through the activation of protein kinase C.     

Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.     

Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15

Recent evidence has suggested that human cyclin-dependent kinase 2 (CDK2) is an essential regulator of cell cycle progression through S phase. CDK2 is known to complex with at least two distinct human cyclins, E and A. The kinase activity of these complexes peaks in G1 and S phase, respectively. The vertebrate CDC2/cyclin B1 complex is an essential regulator of the onset of mitosis and is inhibited by phosphorylation of CDC2 on Thr-14 and Tyr-15. In vitro, CDC2/cyclin B1 is activated by treatment with the members of the Cdc25 family of phosphatases. We found that, like CDC2, CDK2 is also phosphorylated on Thr-14 and Tyr-15 and that treatment of cyclin A or cyclin E immunoprecipitates with bacterially expressed Cdc25M2 (the mouse homolog of human CDC25B) increased the histone H1 kinase activity of these immune complexes 5- to 10-fold. Tryptic peptide mapping demonstrated that Cdc25M2 treatment of cyclin A or cyclin B1 immune complexes resulted in the specific dephosphorylation of Thr-14 and Tyr-15 on CDK2 or CDC2, respectively. Thus, we have confirmed that Cdc25 family members comprise a class of dual-specificity phosphatases. Furthermore, our data suggest that the phosphorylation and dephosphorylation of CDKs on Thr-14 and Tyr-15 may regulate not only the G2/M transition but also other transitions in the cell cycle and that individual cdc25 family members may regulate distinct cell cycle checkpoints.     

Serine 13 is the site of mitotic phosphorylation of human thymidine kinase

It has been reported that the polypeptide of thymidine kinase type 1 (TK1) from human and mouse cells can be modified by phosphorylation. Our laboratory has further shown that the level of human TK phosphorylation increases during mitotic arrest in different cell types (Chang, Z.-F., Huang, D.-Y., and Hsue, N.-C. (1994) J. Biol. Chem. 269:21249-21254). In the present study, we demonstrated that a mutation converting Ser13 to Ala abolished the mitotic phosphorylation of native TK1 expressed in Ltk- cells. Furthermore, we expressed recombinant proteins of wild-type and mutated human TK1 with fused FLAG epitope in HeLa cells, and confirmed the occurrence of mitotic phosphorylation on Ser13 of hTK1. By using an in vitro phosphorylation assay, it was shown that wild-type hTK1, but not mutant TK1(Ala13), could serve as a good substrate for Cdc2 or Cdk2 kinase. Coexpression of p21(waf1/cip1), which is a universal inhibitor of Cdk kinases, in Ltk- fibroblasts also suppressed mitotic phosphorylation of hTK1 expressed in this cell line. Thus, Cdc2 or related kinase(s) is probably involved in mitotic phosphorylation on Ser13 of the hTK1 polypeptide. We also found that mutation on Ser13 did not affect the functional activity of hTK1. As the sequences around Ser13 are highly conserved in vertebrate TK1s, we speculate that phosphorylation of Ser13 may play a role in the regulation of TK1 expression in the cell cycle.     

A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea

By incubating at 30 degrees C in the presence of an energy source, p34(cdc2)/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40 degrees C, the nonpermissive temperature (Nishimoto, T. , E. Eilen, and C. Basilico. 1978. Cell. 15:475-483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34(cdc2)/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34(cdc2)/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40 degrees C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34(cdc2)/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34(cdc2)/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34(cdc2)/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34(cdc2)/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34(cdc2)/cyclin B, which initiates mitosis through the activation of Cdc25C.     

The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis.     

Control of mitotic events by the Cdc42 GTPase, the Clb2 cyclin and a member of the PAK kinase family

BACKGROUND: Cyclins and cyclin-dependent kinases induce and coordinate the events of the cell cycle, although the mechanisms by which they do so remain largely unknown. In budding yeast, a pathway used by the Clb2 cyclin to control bud growth during mitosis provides a good model system in which to understand how cyclin-dependent kinases control cell-cycle events. In this pathway, Clb2 initiates a series of events that lead to the mitosis-specific activation of the Gin4 protein kinase. A protein called Nap1 is required in vivo for the activation of Gin4, and is able to bind to both Gin4 and Clb2. We have used a simple genetic screen to identify additional proteins that function in this pathway. RESULTS: We have found that the Cdc42 GTPase and a member of the PAK kinase family called Cla4 both function in the pathway used by Clb2 to control bud growth during mitosis. Cdc42 and Cla4 interact genetically with Gin4 and Nap1, and both are required in vivo for the mitosis-specific activation of the Gin4 kinase. Furthermore, Cla4 undergoes a dramatic hyperphosphorylation in response to the combined activity of Nap1, the Clb2-Cdc28 kinase complex, and the GTP-bound form of Cdc42. Evidence is presented which suggests that the hyperphosphorylated form of Cla4 is responsible for relaying the signal to activate Gin4. CONCLUSIONS: Previous studies have suggested that cyclin-dependent kinases control the cell cycle by directly phosphorylating proteins involved in specific events, such as nuclear lamins, microtubule-associated proteins and histones. In contrast, our results demonstrate that the Clb2-Cdc28 cyclin-dependent kinase complex controls specific cell-cycle events through a pathway that involves a GTPase and at least two different kinases. This suggests that cyclin-dependent kinases may control many cell-cycle events through GTPase-linked signaling pathways that resemble the intricate signaling pathways known to control many other cellular events.     

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line

It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.     

Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein

DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Here we show that Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage.     

DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site

OBJECTIVE: To describe the comparative impact of current and preventive treatments on incidence of choroidal neovascularization (CNV) and severe vision loss in patients with bilateral soft drusen (BSD). DESIGN: Stochastic model. SETTING: US population. PATIENTS: Prevalence cohort of white patients 43 years or older with BSD. INTERVENTIONS: Application of prophylaxis of 10% to 50% efficacy to 1 or both eyes of patients with BSD, application of laser photocoagulation to eligible CNV lesions, or both. MAIN OUTCOME MEASURES: Proportion of patients with BSD after 10 years with unilateral and bilateral CNV and resultant unilateral and bilateral vision loss to visual acuity of 20/200 or worse. RESULTS: The natural history of patients with BSD generated by the model shows that 12.40% of these patients develop either unilateral or bilateral CNV within 10 years of their entry into the BSD prevalence cohort. Bilateral disease occurs in 3.86% of patients with BSD within 10 years. The proportion of patients with BSD becoming legally blind from CNV within 10 years is 2.54% if no treatment is performed. Current laser treatment for CNV decreases the proportion with legal blindness within 10 years to 2.24%. The addition of a preventive treatment of 10% efficacy applied bilaterally to the current laser treatment regimen decreases the proportion with legal blindness to 1.86%; a 25% effective preventive treatment decreases it to 1.34%. Comparatively, preventive treatment of 10% and 25% efficacy given to the fellow eye only after the first eye has developed CNV decreases the proportion of legally blind patients at 10 years only to 2.06% and 1.77%, respectively. All outcomes vary with sex and age at entry into the BSD cohort. CONCLUSIONS: Patients with BSD face a 12.40% risk of developing CNV within 10 years. The addition of even a modest (10% effective) bilateral preventive treatment to the current regimen for CNV would more than double the prevention of legal blindness in the BSD population relative to current laser treatment; a preventive treatment of 33% efficacy more than halves the rate of legal blindness caused by CNV. Preventive treatment given to the fellow eye only after the first develops CNV has substantially less impact.     

Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation

Treatment of cultured type-1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half-maximal concentrations of thrombin required for each response are 500 and 2 pM, respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half-maximal concentrations of 30 microM for an increase in cell number and DNA synthesis and 100 nM for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10-fold greater than those required for half-maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55-65, 106, 110-115, and 120-130 kDa. The response was rapid (< 1 min) and transient with a peak response after approximately 2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin- or SFLLRNP-mediated reversal of stellation at concentrations of up to 1 microM. In contrast, 1 microM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 microM herbimycin A corresponded to inhibition of receptor-induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor-activated tyrosine kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulation-dependent dephosphorylation in nerve terminals

Clathrin-mediated endocytosis was shown to be arrested in mitosis due to a block in the invagination of clathrin-coated pits. A Xenopus mitotic phosphoprotein, MP90, is very similar to an abundant mammalian nerve terminal protein, epsin, which binds the Eps15 homology (EH) domain of Eps15 and the alpha-adaptin subunit of the clathrin adaptor AP-2. We show here that both rat epsin and Eps15 are mitotic phosphoproteins and that their mitotic phosphorylation inhibits binding to the appendage domain of alpha-adaptin. Both epsin and Eps15, like other cytosolic components of the synaptic vesicle endocytic machinery, undergo constitutive phosphorylation and depolarization-dependent dephosphorylation in nerve terminals. Furthermore, their binding to AP-2 in brain extracts is enhanced by dephosphorylation. Epsin together with Eps15 was proposed to assist the clathrin coat in its dynamic rearrangements during the invagination/fission reactions. Their mitotic phosphorylation may be one of the mechanisms by which the invagination of clathrin-coated pits is blocked in mitosis and their stimulation-dependent dephosphorylation at synapses may contribute to the compensatory burst of endocytosis after a secretory stimulus.     

Phosphorylation of alphaB-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation

The immunofluorescence localization of alphaB-crystallin in U373 MG human glioma cells with an antibody specific for alphaB-crystallin that had been phosphorylated at Ser-45 revealed an intense staining of cells in the mitotic phase of the cell cycle. Phosphorylated forms of alphaB-crystallin in mitotic cells were detected in all cell lines examined and in tissue sections of mouse embryos. Increases in the levels of alphaB-crystallin that had been phosphorylated at Ser-45 and Ser-19, but not at Ser-59, were detected biochemically by isoelectric focusing or SDS-polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of cells collected at the mitotic phase. When we estimated the phosphorylation activity specific for alphaB-crystallin in extracts of mitotic U373 MG cells, using the amino-terminal 72-amino acid peptide derived from unphosphorylated alphaB2-crystallin as the substrate, we found that the activities responsible for the phosphorylation of Ser-45 and Ser-19 were markedly enhanced but that the activity responsible for the phosphorylation of Ser-59 was suppressed. The protein kinases responsible for the phosphorylation of Ser-45 and Ser-59 in the amino-terminal 72-amino acid peptide were partially purified from extracts of cells that had been stimulated by exposure to H2O2 in the presence of calyculin A. The activities responsible for the phosphorylation of Ser-45 and Ser-59 were eluted separately from a column of Superdex 200 at fractions corresponding to about 40 and 60 kDa, respectively, while the kinase for Ser-19 was unstable. p44/42 mitogen-activated protein (MAP) kinase and MAP kinase-activated protein (MAPKAP) kinase-2 were concentrated in the Ser-45 kinase fraction and Ser-59 kinase fraction, respectively. Recombinant human p44 MAP kinase and MAPKAP kinase-2 purified from rabbit muscle selectively phosphorylated Ser-45 and -59, respectively. The Ser-45 kinase fraction and Ser-59 kinase fraction phosphorylated myelin basic protein and hsp27, respectively. These results suggest that the phosphorylations of Ser-45 and Ser-59 in alphaB-crystallin are catalyzed by p44/42 MAP kinase and MAPKAP kinase-2, respectively, in cells and that the phosphorylation of Ser-45 by p44/42 MAP kinase is enhanced while the phosphorylation of Ser-59 by MAPKAP kinase-2 is suppressed during cell division.