Protein Kinase P90 Rsk Research Articles

Metaphase Arrest by Cyclin E-Cdk2 Requires the Spindle-Checkpoint Kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization.

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.

The mechanism of CSF arrest in vertebrate oocytes

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes cytostatic factor (CSF) activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase that activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90 Rsk. Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90 Rsk provides a link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.

Cell cycle transitions in early Xenopus development.

Xenopus oocytes and embryos undergo two major maternally controlled cell-cycle transitions: oocyte maturation and the mid-blastula transition (MBT). During maturation, the essential order of events in the cell cycle is perturbed in that the M phases of Meiosis I and II occur consecutively without an intervening S phase. Use of U0126, a new potent inhibitor of MAPK kinase (MEK), shows that MAPK activation is essential to inhibit the anaphase-promoting complex and cyclin B degradation at the MI/MII transition. If MAPK is inactivated, cyclin B is degraded, S phase commences and meiotic spindles do not form. These events are restored in U0126-treated oocytes by a constitutively active form of the protein kinase p90Rsk. Thus all actions of MAPK during maturation are mediated solely by activation of p90Rsk. At the MBT, commencing with the 13th cleavage division, there are profound changes in the cell cycle. MBT events such as maternal cyclin E degradation and sensitivity to apoptosis are regulated by a developmental timer insensitive to inhibition of DNA, RNA or protein synthesis. Other events, such as zygotic transcription and the DNA replication checkpoint, are controlled by the nuclear:cytoplasmic ratio. Lengthening of the cell cycle at the MBT is caused by increased Tyr15 phosphorylation of Cdc2 resulting from degradation of the maternal phosphatase Cdc25A and continued expression of maternal Wee1. Ionizing radiation causes activation of a checkpoint mediating apoptosis when administered before but not after the MBT. Resistance to apoptosis is associated with increased p27Xic1, the relative fraction of Bcl-2 or Bax in pro- versus anti-apoptotic complexes, and the activity of the protein kinase Akt.

If you take a RiSK, you might get arrested!

Cytostatic factor (CSF) was described almost 30 years ago as the activity in vertebrate eggs that restrains the cell cycle in metaphase of meiosis II. Fertilization provides the liberating Ca2+ burst that signals for the arrest to be broken, meiosis to be completed and the mitotic divisions to commence. Within the past decade, Mos, its target MEK 1 and the MEK substrate mitogen-activated protein kinase (MAPK) have been shown to be essential components of CSF. Recently, two labs 1 Bhatt R.R. Ferrell J.E. The protein kinase p90 Rsk as an essential mediator of cytostatic factor activity. Science. 1999; 286: 1362-1365 Crossref PubMed Scopus (149) Google Scholar , 2 Gross S.D. et al. Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90rsk. Science. 1990; 286: 1365-1367 Crossref Scopus (140) Google Scholar have identified members of the p90rsk family of S6 kinases (Rsk-1 and Rsk-2) as MAPK targets that are necessary and sufficient for CSF activity.

Cell cycle and cell survival: Pinpointing Rsks

During development, some neurons undergo cell death if they are deprived of growth factors. Signals that contribute to such growth factor-dependent survival are mediated, at least in part, by activation of mitogen-activated protein kinases (MAPKs), but the crucial target of the MAPKs has been unknown. Bonni et al. report that the protein kinase Rsk2 (a member of the pp90 ribosomal protein S6 kinase family), which is phosphorylated and activated by MAPKs, may mediate the effects on cell survival in cerebellar granule neurons exposed to brain-derived neurotrophic factor. Rsk2 appears to have two ways of influencing cell survival: It phosphorylates and thus suppresses the effects of BAD, a protein that promotes apoptosis. Rsk2 also phosphorylates and activates the transcription factor CREB (cAMP response element binding protein). Activation of CREB may in turn enhance expression of Bcl2, a protein that promotes cell survival. Nebreda and Gavin discuss these findings and those from a pair of reports that define another role of Rsks in the control of the cell cycle. Before fertilization, most vertebrate eggs are at metaphase of meiosis II. Cytostatic factor (CSF) is the name given to an enzymatic activity present in such eggs, which, when injected into dividing embryos, causes mitotic arrest in metaphase. Such "CSF arrest" can be initiated by the protein kinase Mos, which activates the sequential activation of MAPK kinase MEK and the p42 MAPK. The critical target of p42 MAPK has now been identified as the protein kinase p90 Rsk. Bhatt and Ferrell show that depletion of Rsk from Xenopus egg extracts prevents mitotic arrest in response to activated Mos. Gross et al. demonstrate that activation of Rsk requires its phosphorylation by p42 MAPK and the 3-phosphatidylinositol-dependent kinase-1 (PDK-1). They also constructed a constitutively active form of Rsk that caused metaphase arrest when infected into blastomeres of two-cell Xenopus embryos. Activation of Rsk is thus necessary and sufficient to cause CSF arrest. Bonni, A., Brunet, A., West, A.E., Datta, S.R., Takasu, M.A., and Greenberg, M.A. (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286 : 1358-1362. [Abstract] [Full Text] Bhatt, R.R. and Ferrell Jr., J.E. (1999) The protein Kinase p90 Rsk as an essential mediator of cytostatic factor activity. Science 286 : 1362-1365. [Abstract] [Full Text] Gross, S.D., Schwab, M.S., Lewellyn, A.L., and Maller, J.L. (1999) Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk. Science 286 : 1365-1367. [Abstract] [Full Text] Nebreda, A.R. and Gavin, A.-C. (1999) Cell survival demands some Rsk. Science 286 : 1309-1310. [Summary] [Full Text]

The protein kinase p90 rsk as an essential mediator of cytostatic factor activity.

Persistent activation of p42 mitogen-activated protein kinase (p42 MAPK) during mitosis induces a "cytostatic factor" arrest, the arrest responsible for preventing the parthenogenetic activation of unfertilized eggs. The protein kinase p90 Rsk is a substrate of p42 MAPK; thus, the role of p90 Rsk in p42 MAPK-induced mitotic arrest was examined. Xenopus laevis egg extracts immunodepleted of Rsk lost their capacity to undergo mitotic arrest in response to activation of the Mos-MEK-1-p42 MAPK cascade of protein kinases. Replenishing Rsk-depleted extracts with catalytically competent Rsk protein restored the ability of the extracts to undergo mitotic arrest. Rsk appears to be essential for cytostatic factor arrest.

Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk.

Before fertilization, vertebrate eggs are arrested in metaphase of meiosis II by cytostatic factor (CSF), an activity that requires activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate whether CSF arrest is mediated by the protein kinase p90Rsk, which is phosphorylated and activated by MAPK, a constitutively activated (CA) form of Rsk was expressed in Xenopus embryos. Expression of CA Rsk resulted in cleavage arrest, and cytological analysis showed that arrested blastomeres were in M phase with prominent spindles characteristic of meiotic metaphase. Thus, Rsk appears to be the mediator of MAPK-dependent CSF arrest in vertebrate unfertilized eggs.

Metabotropic glutamate receptor-initiated translocation of protein kinase p90rsk to polyribosomes: a possible factor regulating synaptic protein synthesis.

Maintenance of lasting synaptic efficacy changes requires protein synthesis. We report here a mechanism that might influence translation control at the level of the single synapse. Stimulation of metabotropic glutamate receptors in hippocampal slices induces a rapid protein kinase C-dependent translocation of multifunction kinase p90rsk to polyribosomes; concomitantly, there is enhanced phosphorylation of at least six polyribosome binding proteins. Among the polyribosome bound proteins are the p90rsk-activating kinase ERK-2 and a known p90rsk substrate, glycogen synthase kinase 3beta, which regulates translation efficiency via eukaryotic initiation factor 2B. Thus metabotropic glutamate receptor stimulation could induce synaptic activity-dependent translation via translocation of p90rsk to ribosomes.

Mutations in Rsk-2 cause Coffin-Lowry syndrome

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes CSF activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase, which activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90Rsk which leads to inhibition of cyclin B degradation by the anaphase-promoting complex. Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90Rsk provides a potential link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.