The pathway of MAP kinase mediation of CSF arrest in Xenopus 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 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.

A constitutively active form of the protein kinase p90Rsk1 is sufficient to trigger the G2/M transition in Xenopus oocytes.

The protein kinase p90(Rsk) has previously been implicated as a key target of the MAPK pathway during M phase of meiosis II in Xenopus oocytes. To determine whether Rsk is a mediator of MAPK for stimulation of the G(2)/M transition early in meiosis I, we sought to generate a form of Rsk that would be constitutively active in resting, G(2) phase oocytes. Initial studies revealed that an N-terminal truncation of 43 amino acids conferred enhanced specific activity on the enzyme in G(2) phase, and stability was highest if the C terminus was not truncated. The full-length enzyme is known to be activated by phosphorylation at five sites. Two of these sites and flanking residues were replaced with either aspartic or glutamic acid, and Tyr(699) was mutated to alanine. The resulting construct, termed fully activated (FA) Rsk, had constitutive activity in G(2) phase, with a specific activity equivalent to that of wild type Rsk in M phase. In eight independent experiments approximately 45% of oocytes expressing FA-Rsk underwent germinal vesicle breakdown (GVBD, the G(2)/M transition) in the absence of progesterone, and this effect could be observed even in the presence of the MAPK kinase inhibitor U0126. Moreover, the specific activity of FA-Rsk in vivo was unaffected by U0126. In oocytes that did not undergo GVBD with FA-Rsk expression, subsequent treatment with progesterone resulted in a very rapid rate of GVBD even in the presence of U0126 to inhibit the endogenous MAPK/Rsk pathway. These results indicate that Rsk is the mediator of MAPK effects for the G(2)/M transition in meiosis I and in a subpopulation of oocytes Rsk is sufficient to trigger the G(2)/M transition.

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.

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation.

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk).

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.

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.

The casein kinase Ialpha isoform is both physically positioned and functionally competent to regulate multiple events of mRNA metabolism.

Casein kinase I is a highly conserved family of serine/threonine protein kinases present in every organism tested from yeast to humans. To date, little is known about the function of the higher eukaryotic isoforms in this family. The CKI isoforms in Saccharomyces cerevisiae, however, have been genetically linked to the regulation of DNA repair, cell cycle progression and cytokinesis. It has also been established that the nuclear localization of two of these isoforms is essential for their function. The work presented here demonstrates that the higher eukaryotic CKIalpha isoform is also present within nuclei of certain established cell lines and associated with discrete nuclear structures. The nature of its nuclear localization was characterized. In this regard, CKIalpha was shown to colocalize with factors involved in pre-mRNA splicing at nuclear speckles and that its association with these structures exhibited several biochemical properties in common with known splicing factors. The kinase was also shown to be associated with a complex that contained certain splicing factors. Finally, in vitro, CKIalpha was shown to be capable of phosphorylating particular splicing factors within a region rich in serine/arginine dipeptide repeat motifs suggesting that it has both the opportunity and the capacity to regulate one or more steps of mRNA metabolism.

Casein kinase I: spatial organization and positioning of a multifunctional protein kinase family.

The casein kinase I family of serine/threonine protein kinases is highly conserved from yeast to humans. Until only recently, both the function and regulation of these enzymes remained poorly uncharacterised in that they appeared to be constitutively active and were capable of phosphorylating an untold number of other proteins. While relatively little was known regarding the exact function of the higher eukaryotic isoforms, the casein kinase I (CKI) isoforms from yeast have been genetically linked to vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. All five S. cerevisiae isoforms are known to be associated with discrete cellular compartments and this localization has been shown to be absolutely essential for their respective functions. New evidence now suggests that the CKI isoforms in more complex systems also exhibit non-homogeneous subcellular distributions that may prove vital to defining the function and regulation of these enzymes. In particular, CKIalpha, the most-characterized vertebrate isoform, is associated with cytosolic vesicles, the mitotic spindle and structures within the nucleus. Functions associated with these localizations coincide with those previously reported in yeast, suggesting a conservation of function. Other reports have indicated that each of the remaining CKI isoforms have the capacity to make associations with components of several signal transduction pathways, thereby channeling CKI function toward specific regulatory events. This review will examine what is now known about the higher eukaryotic CKI family members from the perspective localization as a means of gaining a better understanding of the function and regulation of these kinases.

A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte.

Casein kinase I is a family of serine/threonine protein kinases common to all eukaryotes. In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA repair and cell division. In addition, their subcellular localization to membraneous structures and the nucleus is essential for function. In higher eukaryotes, there exist seven genetically distinct isoforms: (alpha), ss, (gamma)1, (gamma)2, (gamma)3, (delta) and (epsilon). Casein kinase I(alpha) exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular structures and the nucleus during interphase, and the spindle during mitosis. casein kinase I has also been shown to modulate critical regulators of growth and DNA synthesis/repair in mammalian cells such as SV40 large T antigen and p53. These results suggest that casein kinase I may be involved in processes similar to those ascribed to the yeast casein kinase I homologues. To define a role for casein kinase I(alpha) in cell cycle regulation, the mouse oocyte was utilized because of its well-defined cell cycle and ease of micromanipulation. Immunofluorescence studies from meiosis I of maturation to the first zygotic cleavage demonstrated that the kinase was associated with structures similar to those previously reported. Microinjection of casein kinase I(alpha) antibodies at metaphase II-arrest and G2 phase, had no effect on the completion of second meiosis or first division. However, microinjection of these antibodies during the early pronucleate phase prior to S-phase onset blocked uptake of the kinase into pronuclei and interfered with proper and timely cell cycle progression to first cleavage. These results suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.

Casein kinase I alpha and alpha L: alternative splicing-generated kinases exhibit different catalytic properties.

Casein kinase I (CKI) is a family of serine/threonine protein kinases found in all eukaryotes examined to date. Here, the rat CKI isoforms alpha and alpha L were cloned and expressed in both eukaryotic and prokaryotic systems. Characterization of the genomic DNA flanking the exon unique to CKI alpha L demonstrated that CKI alpha and CKI alpha L arise by the alternative splicing of a common pre-mRNA molecule. To the best of our knowledge, the alpha L isoform is the only known active serine/threonine kinase to contain an insert within its catalytic domain. Tissue distribution of each splicing isoform was examined by RT-PCR, immunoprecipitation, and Western blotting. Both isoforms were expressed in all tissues tested but at different levels. Bacterially expressed CKI alpha isoforms were active and therefore biochemically characterized. CKI alpha and CKI alpha L proteins were demonstrated to have casein kinase I catalytic properties. More importantly, the recombinant isoform proteins exhibited differences in binding and activity toward common CKI substrates. These observations demonstrate that the alpha L insert within the kinase domain modulates substrate kinetics. These kinetic differences suggest that CKI alpha and CKI alpha L may perform different biological roles.

A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins.

In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha-CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion.

Cell cycle-dependent localization of casein kinase I to mitotic spindles.

Casein kinase I (CKI) is a class of protein kinases ubiquitous to all eukaryotic cells. Recently, cDNA clones encoding several bovine CKI isoforms have been sequenced that show high sequence identity to the HRR25 gene product of the budding yeast Saccharomyces cerevisiae; HRR25 is required for normal cellular growth, nuclear segregation, DNA repair, and meiosis. We have raised polyclonal antibodies to a human erythroid 34-kDa CKI and have sequenced a portion of this kinase. The amino acid sequence identifies the CKI as the alpha-CKI isoform, which is 62% identical to the HRR25 protein kinase. By use of immunofluorescence, the alpha-CKI has been localized to vesicular cytosolic structures and to the centrosome in interphase cells. As cells progress into mitosis, centrospheric staining increases and, in mitosis, alpha-CKI associates with kinetochore fibers. This localization suggests that alpha-CKI, like HRR25, plays a role in the segregation of chromosomes during mitosis and may be cell cycle-regulated both in humans and in yeast.

Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment.

DBA mice were chronically treated with nicotine by continuous intravenous infusion of 4.0 mg/kg/hr for 10 d. Drug-treated mice were tolerant to the acute effects of nicotine on locomotor activity and body temperature. The effect of chronic treatment on the amount of L-3H-nicotine binding and RNA encoding for alpha 4, the most widely expressed nicotinic alpha-subunit, was measured in three brain regions. Chronic treatment increased L-3H-nicotine binding in cortex and midbrain but had no effect in cerebellum. In contrast, chronic treatment had no effect on the levels of mRNA encoding for alpha 4 in any of the three brain regions. Subsequently brains were sectioned and L-3H-nicotine binding was measured using quantitative autoradiographic methods. In addition, the relative amounts of mRNA for the major nicotinic receptor subunits (alpha 4 and beta 2), as well as for three additional minor subunits (alpha 2, alpha 3, and alpha 5), were determined by in situ hybridization histochemistry followed by quantitation of image intensity. Chronic nicotine treatment resulted in increases in the amount of L-3H-nicotine binding in many but not all brain areas measured. In contrast, chronic treatment had little effect on the intensity of the hybridization signal for the nicotinic subunit mRNA. The results suggest that chronic treatment with nicotine under conditions resulting in maximal steady-state increases in L-3H-nicotine binding has little effect on RNA levels encoding any of four nicotinic alpha-subunits and the beta 2-subunit.

An autoradiographic analysis of cholinergic receptors in mouse brain after chronic nicotine treatment.

Quantitative autoradiographic procedures were used to examine the effects of chronic nicotine infusion on the number of central nervous system nicotinic cholinergic receptors. Female DBA mice were implanted with jugular cannulas and infused with saline or various doses of nicotine (0.25, 0.5, 1.0 or 2.0 mg/kg/hr) for 10 days. The animals were then sacrificed and the brains were removed and frozen in isopentane. Cryostat sections were collected and prepared for autoradiographic procedures as previously described. Nicotinic cholinergic receptors were labeled with L-[3H]nicotine or alpha-[125I]bungarotoxin; [3H]quinuclidinyl benzilate was used to measure muscarinic cholinergic receptor binding. Chronic nicotine infusion increased the number of sites labeled by [3H]nicotine in most brain areas. However, the extent of the increase in binding as well as the dose-response curves for the increase were widely different among brain regions. After the highest treatment dose, binding was increased in 67 of 86 regions measured. Septal and thalamic regions were most resistant to change. Nicotinic binding measured by alpha-[125I]bungarotoxin also increased after chronic treatment, but in a less robust fashion. At the highest treatment dose, only 26 of 80 regions were significantly changes. Muscarinic binding was not altered after chronic nicotine treatment. These data suggest that brain regions are not equivalent in the mechanisms that regulate alterations in nicotinic cholinergic receptor binding after chronic nicotine treatment.