B lymphocyte activation is accompanied by phosphorylation of a 72-kDa protein-tyrosine kinase

The cross-linking of membrane IgM on the surface of splenic B lymphocytes or WEHI 231 cells leads to the rapid phosphorylation on tyrosine of a 72-kDa protein as detected in Western blotting experiments using anti-phosphotyrosine antibodies. The 72-kDa phosphoprotein detected in this manner comigrates, in both one- and two-dimensional polyacrylamide gel electrophoresis systems, with PTK72, a 72-kDa protein-tyrosine kinase characterized previously in this laboratory (Zioncheck, T. F., Harrison, M. L., Isaacson, C. C., and Geahlen, R. L. (1988) J. Biol. Chem. 263, 19195-19202). Anti-phosphotyrosine antibodies and anti-PTK72 antibodies immunoprecipitate the same protein-tyrosine kinase from extracts of anti-IgM-activated cells as determined by immune complex kinase assays and one-dimensional phosphopeptide mapping. These results indicate that the tyrosine phosphorylation of a 72-kDa protein-tyrosine kinase is an early event in the activation of B lymphocytes via the antigen receptor.     

Regulation of binding of lamin B receptor to chromatin by SR protein kinase and cdc2 kinase in Xenopus egg extracts

Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR protein kinases, abolished this stimulation, suggesting that an SR protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.     

Enhanced JNK activation by NESK without kinase activity upon caspase-mediated cleavage during apoptosis

Nck-interacting kinase-like embryo-specific kinase (NESK) is a protein kinase that is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. NESK belongs to the germinal center kinase (GCK) family and selectively activates the c-Jun N-terminal kinase (JNK) pathway when overexpressed in cultured cells. Some members of the GCK family have been shown to be proteolytically cleaved and activated during apoptosis. Here, we report that NESK is also proteolytically cleaved during apoptosis. Treatment of NESK-transfected HeLa cells with TNF-alpha in the presence of cycloheximide or with staurosporine induced proteolytic cleavage of NESK. The cleavage of NESK occurred at two sites, generating three fragments: an N-terminal fragment containing a kinase domain, an intermediate fragment and a C-terminal fragment containing a regulatory CNH domain. These two cleavages occurred in a stepwise manner and were dependent on a caspase activity. The cleavage sites were identified as aspartic acid residues at 868 and 1091. The N-terminal fragment had less kinase activity than the full-length NESK and did not activate the JNK pathway. In contrast, the C-terminal fragment activated the JNK pathway more strongly than the full-length NESK and promoted TNF-alpha-induced apoptotic cell death. These results implicate NESK in the JNK pathway-mediated promotion of apoptosis through its C-terminal regulatory domain generated by proteolytic cleavage during apoptosis, in a unique manner different from other GCK family kinases.     

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.     

Germinal vesicle material is dispensable for oscillations in cdc2 and MAP kinase activities,cyclin B degradation and synthesis during meiosis in Xenopus oocytes

We have investigated at a molecular level the requirements for germinal vesicle (nuclear) material during the course of meiosis in Xenopus oocytes. We present the localization of some cell cycle proteins in stage VI oocytes; most of those analyzed are cytoplasmic, although some (MAD, 26S proteasome) are distributed between the cytoplasm and the germinal vesicle. By analyzing changes in individual oocytes, we find that the unphosphorylated form of cyclin B2 disappears and the phosphorylated form is then degraded in both nucleated and enucleated oocytes. Enucleated oocytes are also capable of resynthesizing both cyclin B1 and cyclin B2 after the initial degradation and of reactivating cdc2 kinase. Synthesis of mos protein and activation of MAP kinase concomitant with cdc2-cyclin B reactivation are also unaffected by prior removal of the germinal vesicle.     

The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase

Entry into mitosis by mammalian cells is triggered by the activation of the cdc2/cyclin B holoenzyme. This is accomplished by the specific dephosphorylation of key residues by the cdc25C phosphatase. The polo-like kinases are a family of serine/threonine kinases which are also implicated in the control of mitotic events, but their exact regulatory mechanism is not known. Recently, a Xenopus homologue, PLX1, was reported to phosphorylate and activate cdc25, leading to activation of cdc2/cyclin B. Jurkat T leukemia cells were chemically arrested and used to verify that PLK protein expression and its phosphorylation state is regulated with respect to cell cycle phase (i.e., protein is undetectable at G1/S, accumulates at S phase and is modified at G2/M). Herein, we show for the first time that endogenous human PLK protein immunoprecipitated from the G2/M-arrested Jurkat cells directly phosphorylates human cdc25C. In addition, we demonstrate that recombinant human (rh) PLK also phosphorylates rhcdc25C in a time- and concentration-dependent manner. Phosphorylation of endogenous cdc25C and recombinant cdc25C by PLK resulted in the activation of the phosphatase as assessed by dephosphorylation of cdc2/cyclin B. These data are the first to demonstrate that human PLK is capable of phosphorylating and positively regulating human cdc25C activity, allowing cdc25C to dephosphorylate inactive cdc2/cyclin B. As this event is required for cell cycle progression, we define at least one key regulatory mode of action for human PLK in the initiation of mitosis.     

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.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.     

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.     

Clofibrate-induced apoptosis is mediated by Ca2+-dependent caspase-12 activation

The mechanism of fibrate-induced myopathy was investigated in this report. When clofibrate (30 to 300 microM) was applied to L6 rat skeletal myoblasts, dose-dependently apoptosis was observed within 24 h. In the apoptotic myoblasts, a caspase-12 cleavage was observed at 2 h and with following caspases-9 and -3-related cascade activation. In contrast, the neutral protease calpain, that is a key enzyme in ER stress-related apoptosis via caspase-12 activation, was significantly decreased during apoptosis. Next, the authors evaluated a role of calcium-dependent signal(s). When clofibrate was added into medium, cytosolic calcium concentration was rapidly and persistently increased. On the other hand, an addition of 10 mM EGTA depressed sustained calcium phase, and concurrent myoblasts apoptosis was completely inhibited. Taken together, our findings indicate that the clofibrate-induced myopathy is triggered by Ca2+ influx, then activated cytosolic caspase-12 through calpain-independent cascade, and consequently caused apoptotic DNA fragmentation.     

Activation of p34cdc2 kinase by cyclin A

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.     

Immortal, non-tumourigenic mouse mammary outgrowths express high levels of cyclin B1 and activation of cyclin B1/cdc2 kinase

Abstract. Neoplastic transformation of mouse mammary epithelial cells is the result of several identifiable phenotypic changes which presumably require sequential genetic alterations. In our model system, mammary cells progress from a mortal state (virgin duct) to several morphologically distinct intermediate states. The intermediate states are distinct cell populations that are phenotypically identified as immortal, non-tumourigenic (i.e. EL11), weakly tumourigenic ductal/alveolar hyperplasia (i.e. EL12) and moderately tumourigenic alveolar hyperplasiaa (i.e. TM12) to invasive tumours (i.e. EL12T/TM12T). We have studied the changes in total cyclin A and B1 levels, cyclin A and B1 complexed to cdc2, cyclin B1cdc2 kinase activity and cyclin D proteins in EL11 and EL12 immortalized outgrowth lines. Results revealed increased levels in total cyclin B1(> 5-fold), cyclin B1/cdc2 (3–4-fold) and cyclin B1/cdc2 kinase activity (2–3.5-fold) in EL11 and EL12 phenotypes when compared to control mammary gland (virgin). No changes in the levels of total cyclin A or cycln A associated to cdc2 were observed. Cyclin D1, D2 and D3 protein levels were low in the EL11 immortal ductal outgrowth. Exposure to hormones via a pituitary isograft stimulated the synthesis of cyclin D1 and D2 but not D3 associated to cdk4 as well as total cdk4 proteins. Bromodeoxyuridine (BrdUrd) labelling indices showed marked increases in immortal ductal outgrowths (EL11 and EL12) when compared to virgin, suggesting that epithelial cells are cycling in these cell populations. Even in the presence of hormone stimulation, EL11 outgrowths were not tumourigenic, suggesting that other events are necessary to drive the cells to a tumourigenic phenotype. The results suggest that increased levels of cyclin B1 and cyclin B1-cdc2 kinase activities are early events and may be an important marker for the immortalized phenotype.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Inhibition of cdc2 activation by INH/PP2A.

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. We first show that INH is a conventional form of PP2A with a B alpha regulatory subunit. We next explore the mechanism by which it inhibits cdc2 activation by examining the effect of purified PP2A on the reaction pathways controlling cdc2 activity. Our results suggest that although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, our results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis.     

Protein kinase Cepsilon binds peripherin and induces its aggregation, which is accompanied by apoptosis of neuroblastoma cells

A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCepsilon induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCepsilon. PKCepsilon and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCepsilon. The interaction was specific for PKCepsilon since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCeta and -, which pulled down minute amounts. PKCepsilon interacts with vimentin through the same structures but does not induce its aggregation. When the PKCepsilon C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCepsilon through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCepsilon.