Biochemical characterization of the p34cdc2 protein kinase component of purified maturation-promoting factor from Xenopus eggs

Genetic studies in the fission yeast Schizosaccharomyces pombe and biochemical data in oocytes and eggs of Xenopus laevis have implicated the product of the cdc2+ gene as critical for the G2 to M transition in the cell cycle. The product of the cdc2+ gene is a 34-kDa serine/threonine protein kinase, designated p34cdc2, that is a component of purified maturation-promoting factor (MPF) and also of purified mammalian growth-associated histone H1 kinase. The biochemical properties of p34cdc2 H1 kinase activity in the MPF complex were studied. Phosphorylation of the p45cyclin component in the MPF complex by p34cdc2 exhibited kinetics consistent with an intramolecular reaction. On glycerol gradient centrifugation, MPF kinase against several substrates sedimented with an apparent Mr = 45,000-55,000. p34cdc2 was found to utilize ATP, GTP, and adenosine 5'-O-(3-thiotriphosphate) with apparent Km values of 75, 700, and 250 microM, respectively. The kinase activity was inhibited by beta-glycerophosphate, NaF, and zinc, whereas p-nitrophenyl phosphate was slightly stimulatory. The relative rates of phosphorylation of various substrates by MPF and growth-associated H1 kinase were similar. These findings should prove useful in further work on the regulation of MPF kinase activity and characterization of its substrates.     

INH, a negative regulator of MPF, is a form of protein phosphatase 2A.

MPF, a protein kinase complex consisting of cyclin and p34cdc2 subunits, promotes the G2 to M phase transition in eukaryotic cells. The pathway of activation and inactivation of MPF is not well understood, although there is strong evidence that removal of phosphate from a tyrosine residue on p34cdc2 is part of the activation process. INH was originally identified as an activity that could inhibit the posttranslational activation of a latent form of MPF, called pre-MPF, in immature (G2 phase-arrested) Xenopus oocytes. We have purified INH and demonstrated that it is a form of protein phosphatase 2A. Both INH and the catalytic subunit of protein phosphatase 2A can directly inactivate an isolated p34cdc2-cyclin complex. Both cyclin and p34cdc2 become dephosphorylated; the rate of inactivation closely parallels the removal of phosphate from a specific site on p34cdc2. We propose that INH opposes MPF activation by reversing this critical phosphorylation.     

Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation

MPF extracted from starfish oocytes copurifies with an M phase-specific H1 histone kinase encoded by a homolog of the fission yeast cell cycle control gene cdc2+. The most purified preparations contain p34cdc2 as the only major protein. Activation of the p34cdc2 kinase is correlated with appearance of the MPF activity both in vivo and in vitro. The increase in protein kinase activity is associated with p34cdc2 dephosphorylation and the decrease in protein kinase activity on leaving M phase with rephosphorylation. Microinjection of a peptide perfectly conserved in p34cdc2 from yeast to humans induces meiotic maturation, suggesting that an inhibitory component in G2 arrested oocytes interacts with this region of the p34cdc2 kinase. We propose that initiation of M phase is brought about by the dephosphorylation of p34cdc2, leading to increase in its protein kinase activity.     

Xenopus oocytes and the biochemistry of cell division

The control of cell proliferation involves both regulatory events initiated at the plasma membrane that control reentry into the cell cycle and intracellular biochemical changes that direct the process of cell division itself. Both of these aspects of cell growth control can be studied in Xenopus oocytes undergoing meiotic maturation in response to mitogenic stimulation. All mitogenic signaling pathways so far identified lead to the phosphorylation of ribosomal protein S6 on serine residues, and the biochemistry of this event has been investigated. Insulin and other mitogens activate ribosomal protein S6 kinase II, which has been cloned and sequences in oocytes and other cells. This enzyme is activated by phosphorylation on serine and threonine residues by an insulin-stimulated protein kinase known as MAP-2 kinase. MAP kinase itself is also activated by direct phosphorylation on threonine and tyrosine residues in vivo. These results reconstitute one step of the insulin signaling pathway evident shortly after insulin receptor binding at the membrane. Several hours after mitogenic stimulation, a cell cycle cytoplasmic control element is activated that is sufficient to cause entry into M phase. This control element, known as maturation-promoting factor or MPF, has been purified to near homogeneity and shown to consist of a complex between p34cdc2 protein kinase and cyclin B2. In addition to apparent phosphorylation of cyclin, regulation of MPF activity involves synthesis of the cyclin subunit and its periodic degradation at the metaphase----anaphase transition. The p34cdc2 kinase subunit is regulated by phosphorylation/dephosphorylation on threonine and tyrosine residues, being inactive when phosphorylated and active when dephosphorylated. Analysis of phosphorylation sides in histone H1 for p34cdc2 has revealed a consensus sequence of (K/R)S/TP(X)K/R, where the elements in parentheses are present in some but not all sites. Sites with such a consensus are specifically phosphorylated in mitosis and by MPF in the protooncogene pp60c-src. These results provide a link between cell cycle control and cell growth control and suggest that changes in cell adhesion and the cytoskeleton in mitosis may be regulated indirectly by MPF via protooncogene activation. S6 kinase II is also activated upon expression of MPF in cells, indicating that MPF is upstream of S6 kinase on the mitogenic signaling pathway. Further study both of the signaling events that lead to MPF activation and of the substrates for phosphorylation by MPF should lead to a comprehensive understanding of the biochemistry of cell division.     

Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone.     

Cyclin is a component of maturation-promoting factor from Xenopus

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.     

An extra copy of nimEcyclinB elevates pre-MPF levels and partially suppresses mutation of nimTcdc25 in Aspergillus nidulans.

Previous work has shown that nimA encodes a cell cycle regulated protein kinase that is required along with the p34cdc2 histone H1 kinase (MPF) for mitosis in Aspergillus nidulans. We have now identified two other gene products required for mitosis in A.nidulans. nimT encodes a protein similar to the fission yeast cdc25 tyrosine phosphatase and is required for the conversion of pre-MPF to MPF and nimE encodes a B-type cyclin which is a subunit of MPF. A new genetic interaction between nimEcyclinB and nimTcdc25 type genes is reported. Increased copy number of nimEcyclinB can suppress mutation of nimTcdc25 and also lead to increased accumulation of tyrosine phosphorylated p34cdc2 (pre-MPF). This biochemical observation suggests an explanation for the genetic complementation. If nimEcyclinB recruits p34cdc2 for tyrosine phosphorylation to form pre-MPF it follows that increased expression of nimEcyclinB would increase the level of pre-MPF. The increased level of pre-MPF generated may then allow the mutant nimTcdc25 protein to convert enough pre-MPF to MPF and thus permit some mitotic progression. We also demonstrate that correct cell cycle regulation by the p34cdc2 protein kinase pathway is essential for correct developmental progression in A.nidulans.     

MPF is activated in growing immature Xenopus oocytes in the absence of detectable tyrosine dephosphorylation of P34cdc2.

Tyrosine-phosphorylated p34cdc2 and cyclin B2 are present and physically associated in small growing stage IV oocytes (800 microns in diameter) of Xenopus laevis. Microinjection of M-phase promoting factor (MPF) into stage IV oocytes induces germinal vesicle breakdown and the activation of the kinase activity of the p34cdc2/cyclin B2 complex measured on p13suc1 beads. During the in vivo activation of MPF in stage IV oocytes, p34cdc2 tyrosine dephosphorylation is not detectable, in contrast to stage VI oocytes. Addition of cycloheximide in MPF-injected stage IV oocytes induces neither the inhibition of histone H1 kinase activity nor the cyclin B2 degradation. Therefore, the activation mechanism of histone H1 kinase in stage IV oocytes does not require detectable tyrosine dephosphorylation of p34cdc2. It is suggested rather that the tyrosine phosphorylation of p34cdc2 plays a role in inhibiting cyclin B2 degradation.     

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.     

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.     

Regulation of maturation-promoting factor by protein kinase C in Chaetopterus oocytes

Summary

We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34^cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34^cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80^cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34^cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.     

Cyclin B targets p34cdc2 for tyrosine phosphorylation.

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34cdc2 subunit and the phosphorylation of the cylin Bcdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34cdc2, using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G2 phase, a massive tyrosine phosphorylation of p34cdc2 occurs. This large p34cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34cdc2 concentration. It even appears to affect only a fraction (non-immunoprecipitable by anti-PSTAIR antibodies) of the total p34cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G2 phase; (ii) both tyrosine-phosphorylated p34cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34cdc2; and (iv) inhibition of cyclin synthesis by emetine inhibits p34cdc2 tyrosine phosphorylation without affecting the p34cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.     

Maturation/M-phase promoting factor regulates aging of porcine oocytes matured in vitro

Control of oocyte aging during manipulation of matured oocytes should have advantages for recently developed reproductive technologies, such as cloning after nuclear transfer. We have shown that the enhanced activation ability and fragmentation of porcine in vitro matured and aged oocytes bore a close relationship to the gradual decrease in maturation/M-phase promoting factor (MPF) activity and that porcine aged oocytes contained plenty of MPF, but it was in an inactive form, pre-MPF, as a result of phosphorylation of its catalytic subunit p34(cdc2) and, therefore, had low MPF activity. We incubated porcine oocytes with vanadate and caffeine, which affected the phosphorylation status and MPF activity, and evaluated their activation abilities and fragmentation frequencies. Incubation of nonaged oocytes with vanadate increased p34(cdc2) phosphorylation and reduced MPF activity to levels similar to those of aged oocytes and increased their parthenogenetic activation and fragmentation rates compared with those of the control oocytes. Conversely, treating aged oocytes with caffeine reduced p34(cdc2) phosphorylation and increased MPF activity. These oocytes showed significantly lower parthenogenetic activation and fragmentation rates than aged mature oocytes. These results suggest that MPF activity is a key mechanism of oocyte aging and controlling MPF activity by altering p34(cdc2) phosphorylation with these chemicals may enable oocyte aging to be manipulated in vitro. We expect those ideas will be applied practically to pig cloning.     

p34cdc2 kinase-mediated release of lamins from nuclear ghosts is inhibited by cAMP-dependent protein kinase.

During mitosis the lamins are found in a hyperphosphorylated and soluble state. p34cdc2 kinase (MPF), a protein kinase complex with a pivotal role during mitosis, has been found to phosphorylate the lamins and, in some cases, though not all, to cause depolymerization of the lamina in vitro. Due to the variety of protein interactions in the lamina, there is a probable requirement for multiple enzyme activities to effect its breakdown in mitosis. Using nuclear ghosts as substrate, we have fractionated a Xenopus mitotic extract into a lamin-releasing fraction (p34cdc2 kinase) and a fraction that inhibits p34cdc2 kinase-mediated lamin release if the nuclear ghosts are first preincubated in it. The lamin-release-inhibiting activity in the p34cdc2 kinase-depleted mitotic extract is, in turn, inhibited if PKI, a protein kinase inhibitor specific for PKA, is included in the preincubation reaction mixture. Furthermore, a similar degree of inhibition can be achieved by using purified PKA to preincubate the nuclear ghosts. This suggests that dephosphorylation of PKA substrate sites is necessary for lamin depolymerization.     

A novel M phase-specific H1 kinase recognized by the mitosis-specific monoclonal antibody MPM-2

At the onset of mitosis, eukaryotic cells display an abrupt increase in a Ca2(+)- and cyclic nucleotide-independent histone H1 kinase activity, referred to as growth-associated or M phase-specific H1 kinase. The molecular basis for this activity is generally attributed to a kinase complex that consists of the p34cdc2 protein and cyclin, and exhibits maturation-promoting factor (MPF) activity. In the present study, we show that more than one kinase contributes to M phase-specific H1 kinase activity. When mature Xenopus oocyte extract prepared with ATP gamma S and NaF was fractionated by gel filtration, two prominent peaks of H1 kinase activity were detected, with apparent molecular masses of 600 and 150 kDa. The 150-kDa kinase copurified with the p34cdc2 protein and was immobilized by the suc 1 gene product p13 and anti-cyclin B2, which are specific for the cdc2 kinase complex. However, the 600-kDa kinase did not satisfy any of these criteria, thus identifying it as a novel M phase-specific H1 kinase. Only the 600-kDa kinase was recognized by the mitosis-specific monoclonal antibody, MPM-2, which inhibits Xenopus oocyte maturation and immunodepletes MPF activity. Furthermore, not only did the full activation of this kinase (MPM-2 kinase) coincide with the activation of MPF during the cell cycle, but also MPM-2 kinase-positive fractions obtained by gel filtration accelerated progesterone-induced oocyte maturation. It is, therefore, likely that MPM-2 kinase is a positive regulator in the M phase induction pathway.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

Purification and characterization of maturation-promoting factor in fish.

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.     

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.     

Survivin phosphorylation and M-phase promoting factor in oral carcinogenesis

Survivin is a recently described inhibitor of apoptosis and mitotic regulator which is selectively over-expressed in human tumors. Its expression rate is predictive of disease progression, early recurrences and resistance to therapy. Up-regulation of survivin in oral pre-malignant lesions (OPL) and in oral squamous cell carcinoma (OSCC) has already been demonstrated in previous studies. A critical step for activation of survivin has been identified in the phosphorylation on Thr34 by the main mitotic kinase p34cdc2-cyclin B1. The aim of this work was to investigate the relationship between survivin, its phosphorylated active form (p-survivin) and M-phase promoting factor (MPF), p34cdc2-cyclin B1 in oral carcinogenesis. 32 OSCCs and 17 OPLs from surgical specimens were studied for cyclin B1, p-survivin, survivin, and p34cdc2 expression by immunohistochemistry. All cases of OSCC expressed survivin and its expression rate was correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 80% of cases, while p-34cdc2 was over-expressed in all OSCCs. All OPLs associated with OSCC expressed survivin and its levels were correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 70% of cases, while p-34cdc2 was positive in all OPLs. In conclusion, this study demonstrated that MPF, survivin and p-survivin are expressed during early and late phase of oral carcinogenesis. MPF proteins, which are co-expressed on mitotic apparatus, could represent a potential target for therapies based on manipulation of survivin phosphorylation, which would induce apoptosis in cancer cells.     

Regulation of Xenopus p21-activated kinase (X-PAK2) by Cdc42 and maturation-promoting factor controls Xenopus oocyte maturation

Signal transduction cascades involved in regulation of the cell cycle machinery are poorly understood. In the Xenopus oocyte model, meiotic maturation is triggered by MPF, a complex of p34(cdc2)-cyclin B, which is activated in response to a progesterone signal by largely unknown mechanisms. We have previously shown that the p21-activated kinase (PAK) family negatively regulates the MPF amplification loop. In this study, we identify the endogenous PAK2 as a key enzyme in this regulation and describe the pathways by which PAK2 is regulated. We show that the small GTPase Cdc42 is required for maintenance of active endogenous X-PAK2 in resting stage VI oocytes, whereas Rac1 is not involved in this regulation. During the process of maturation, X-PAK2 phosphorylation results in its inactivation and allows maturation to proceed to completion. Activation of mitogen-activated protein kinase and cyclin B-p34(cdc2) is coincident with X-PAK2 inactivation, and purified active MPF inhibits X-PAK2, demonstrating the existence of a new positive feedback loop. Our results confirm and extend the importance of p21-activated kinases in the control of the G(2)/M transition. We hypothesize that the X-PAK2/Cdc42 pathway could link p34(cdc2) activity to the major cytoskeleton rearrangements leading to spindle migration and anchorage to the animal pole cortex.