The internal structure of different alleles of the minisatellite present at the 3′ end of the apolipoprotein B (ApoB) gene has been analysed by different approaches including sequencing. The repeat unit arrangements of the minisatellite on 570 chromosomes belonging to European and African populations were thus determined. It was possible to group the alleles using this structural criterion much more clearly than by the number of repeat units which can in some cases be misleading in case-control genetic epidemiological studies using such DNA sequences as markers. We were thus able to define five types (a to e) of alleles and their subtypes and to recognize clearly those which are, respectively, specific of the African and Caucasian populations. A phylogeny of the different alleles found in all human populations could also be deduced by this approach. The different putative mutational events leading from one type, or subtype, to the other were simply determined as point mutations, expansion/contraction and conversion events. Sequencing of one chimpanzee's allele suggested that the ApoB minisatellite was present before divergence between great apes and humans. It was determined also that a particular ApoB gene haplotype was in linkage disequilibrium with the minisatellite (a) type of alleles. This and the observation that the potential scaffold attachment regions (SAR) and topoisomerase II binding sites present in this minisatellite have a different distribution between the Caucasian and the African specific alleles suggest that the minisatellite could be involved in the epidemiology of coronary diseases.
Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.
Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.
Protein phosphorylation is a post-translational modification essential for the control of the activity of most enzymes in the cell. This protein modification results from a fine-tuned balance between kinases and phosphatases. PP2A is one of the major serine/threonine phosphatases that is involved in the control of a myriad of different signaling cascades. This enzyme, often misregulated in cancer, is considered a tumor suppressor. In this review, we will focus on PP2A-B55, a particular holoenzyme of the family of the PP2A phosphatases whose specific role in cancer development and progression has only recently been highlighted. The discovery of the Greatwall (Gwl)/Arpp19-ENSA cascade, a new pathway specifically controlling PP2A-B55 activity, has been shown to be frequently altered in cancer. Herein, we will review the current knowledge about the mechanisms controlling the formation and the regulation of the activity of this phosphatase and its misregulation in cancer.
X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34cdc2 but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1. X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34cdc2 but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1. In Xenopus laevis, oocytes are naturally arrested at the G2/M transition of meiosis I. Upon release of progesterone by the follicle cells surrounding the oocytes, maturation occurs and the cell cycle resumes. Translation of the proto-oncogene c-Mos (a MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown. kinase kinase), is an essential preliminary event (2Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar, 3Kanki J.P. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5794-5798Crossref PubMed Scopus (71) Google Scholar) in turning on the MAPK cascade, which eventually results in almost simultaneous activation of MAPK and maturation-promoting factor (MPF), a heterodimer composed of p34cdc2 kinase and cyclin B (4Labbé J.C. Capony J.P. Caput D. Cavadore J.C. Derancourt J. Kaghad M. Lelias J.M. Picard A. Dorée M. EMBO J. 1989; 8: 3053-3058Crossref PubMed Scopus (375) Google Scholar, 5Gautier J. Maller J.L. EMBO J. 1991; 10: 177-182Crossref PubMed Scopus (155) Google Scholar). Eggs eventually arrest again at metaphase of second meiosis with high MPF and cytostatic factor (in which c-Mos participates (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar)) activities until fertilization. Conversion of inactive pre-MPF complexes, stored in oocytes, into active MPF requires dephosphorylation of residues Thr14 and Tyr15 on p34cdc2 kinase (7Strausfeld U. Labbé J.C. Fesquet D. Cavadore J.C. Picard A. Sadhu K. Russell P. Dorée M. Nature. 1991; 351: 242-245Crossref PubMed Scopus (443) Google Scholar). Dephosphorylation of these amino acids is crucial for MPF activation, and this process is highly regulated by an activating phosphatase, Cdc25 (for review see Ref. 8Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar), and two inhibitory kinases, Myt1 (9Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (533) Google Scholar) and Wee1 (10Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (268) Google Scholar).Although oocyte maturation has been extensively studied over the past 10 years as a model to understand the mechanisms involved in reentry into the cell cycle, the signal transduction pathway between progesterone binding to its receptor and the activation of the MAPK cascade and MPF is ill understood. Protein kinase A (PKA) appears to be a crucial player in these events, because activation of progesterone receptor is followed by a sudden drop in cAMP concentration (11Speaker M.C. Butcher F.R. Nature. 1977; 267: 848-849Crossref PubMed Scopus (105) Google Scholar) and likely a subsequent inactivation of protein kinase A. Indeed, microinjection of the PKA catalytic subunit prevents progesterone-induced maturation in oocytes, whereas the expression of the PKA regulatory subunit is sufficient to induce maturation (12Maller J. Krebs E. J. Biol. Chem. 1977; 252: 1712-1718Abstract Full Text PDF PubMed Google Scholar). Matten et al. (13Matten W. Daar I. Vande Woude G.F. Mol. Cell. Biol. 1994; 14: 4419-4426Crossref PubMed Scopus (109) Google Scholar) reported that PKA negatively regulates maturation by controlling both c-Mos translation and Cdc25 activation. How PKA acts on c-Mos de novo translation is, however, unclear, and we have demonstrated that in conditions in oocytes in which activation of MAPK is allowed, PKA does not inhibit the c-Mos translation (14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar), raising the possibility that PKA could act on a single target in the negative regulation of maturation.A number of studies demonstrate the importance of the MAPK cascade in MPF activation (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar, 15Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 9: 2131-2138Crossref Scopus (189) Google Scholar) during Xenopus oocyte maturation. Indeed, microinjection of recombinant c-Mos is itself sufficient to induce oocyte maturation in the absence of hormonal treatment (16Yew N. Mellini M.L. Vande Woude G.F. Nature. 1992; 355: 649-652Crossref PubMed Scopus (202) Google Scholar, 17Sagata N. Daar I. Oskarsson M. Showalter S.D. Vande Woude G.F. Science. 1989; 245: 643-646Crossref PubMed Scopus (249) Google Scholar), whereas CL100 (MAPK phosphatase-1) inhibits progesterone-induced maturation (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Important work in Saccharomyces cerevisiaetoward the understanding of the control of the MAPK cascade has been undertaken in the last few years. The response to the mating pheromone signal is mediated by the direct interaction of the Gβ subunit of G protein heterotrimers to Ste20, a serine threonine kinase (19Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-194Crossref PubMed Scopus (183) Google Scholar). Activation of Ste20 results in activation of the MAPK kinase kinase Ste11, which allows the MAPK cascade to be turned on and results in MAPK (Kss1 and Fus3) activation (reviewed in Ref. 20Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 162-167Crossref Scopus (188) Google Scholar). InXenopus oocytes, Gotoh et al. (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) showed that microinjection of constitutively active Ste11 is able to efficiently induce the maturation process. Moreover, addition of the N-terminal regulatory domain of Ste11 to Xenopus extracts prevents a constitutive form of Ste20 from activating MAPK (21Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar).p21-activated kinases (PAKs) are Ste20 homologues. Understanding of their functional importance in signal transduction and in regulation of the actin cytoskeleton is rapidly growing (see Refs. 22Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 23Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Manser E.H.Y. Huang T.H. Loo X.Q. Chen J.M. Dong T. Leung L. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar). We have cloned Xenopus PAKs and recently demonstrated that a member of this family is involved in regulation of the oocyte maturation process (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). Indeed, we showed that microinjection of a constitutive truncated version of X-PAK1 (X-PAK1-Cter) into oocytes completely blocks both insulin and progesterone mediated maturation, and conversely, the dominant negative version of the kinase facilitated this process. In this present report, we focus on how X-PAK1 negatively regulates maturation. Our results demonstrate that X-PAK1-Cter prevents c-Mos synthesis and MAPK activation in progesterone-treated oocytes. Even if MAPK is activated by injection of recombinant c-Mos, no H1 kinase activity can be detected. All the enzymes involved in regulation of p34cdc2 kinase are affected by X-PAK1-Cter expression in progesterone-treated oocytes. We show that in the presence of X-PAK1 kinase activity, pre-MPF complexes can efficiently form but activation of pre-MPF fails. Furthermore, in the presence of active MPF, X-PAK1 kinase activity prevents the amplification loop from being switched on. We have analyzed a possible link of transduction pathways used by PKA and X-PAK in controlling maturation and show that X-PAK1-Cter is still capable of inhibiting maturation induced by the regulatory domain of PKA. Finally, using cell-free Xenopus egg cycling extracts, which mimic the alternation of S and M phases of the first embryonic cycles, we can reproduce the inhibition of entry into mitosis with the catalytic domain of X-PAK1. Thus the involvement of XenopusPAK in G2/M transition is not restricted to oocyte meiotic maturation and can be extended to the first embryonic cell cycles.DISCUSSIONOocyte maturation is a highly regulated process, and a complete understanding of the many intertwining regulations involved is far from being a reality. GVBD is always associated with high cyclin B-p34cdc2 activity, which results from the activation of stockpiled complexes, occurring by a well described amplification loop. Although experimental conditions have been described in which GVBD can occur in the absence of MAPK activity (34Rime H. Haccard O. Ozon R. Dev. Biol. 1992; 151: 105-110Crossref PubMed Scopus (43) Google Scholar), it is widely accepted that the MAPK cascade participates in normal maturation in the activation of MPF. Understanding the event required for activation of the first cyclin B-p34cdc2 complex, which in turn will start the amplification loop, is crucial to explain the maturation process, and in this regard identification of new regulatory proteins will certainly help to reconstitute the sequence of activation of the different pathways.In this report we investigate how constitutive Xenopusp21-activated kinase negatively controls the oocyte maturation process. Our results indicate that X-PAK1 mimics the effects previously reported for the catalytic subunit of protein kinase A by preventing c-Mos accumulation as well as MAPK and MPF activation (13Matten W. Daar I. Vande Woude G.F. Mol. Cell. Biol. 1994; 14: 4419-4426Crossref PubMed Scopus (109) Google Scholar, 14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar). We show, in a context in which MAPK activation is activated by injection of c-Mos, that accumulation of the endogenous proto-oncogene does occur, in the presence of X-PAK1. Thus X-PAK1 does not interfere with the amplification loop in which active MAPK controls c-Mos synthesis (42Matten W.T. Copeland T.D. Ahn N.G. Vande Woude G.F. Dev. Biol. 1996; 179: 485-492Crossref PubMed Scopus (88) Google Scholar) but rather prevents the initial MAPK activation. Even on c-Mos microinjection, no H1 kinase activity is detected, indicating that X-PAK1 also affects MPF activation. We used recombinant cyclin B, and active MPF, to demonstrate that formation of new pre-MPF complexes is not affected but that they are not activated in the presence of active X-PAK1.Indeed, we show that the regulatory kinases and Cdc25 phosphatase identified in MPF amplification during the maturation process remain in their initial state. A well accepted concept is that activation of the first MPF molecules is the result of activation and/or inactivation of Cdc25 and Myt1, respectively, by an upstream regulating element different from MPF itself. Indeed, in SDS-PAGE, Cdc25 displays a complex phosphorylation pattern and can also be phosphorylated and activated by kinases other than MPF itself, Plx-1 (36Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar), and possibly Raf-1(43).Raf-1 may be a Cdc25-activating kinase (43Galaktionov K. Jessus C. Beach D. Genes Dev. 1995; 9: 1046-1058Crossref PubMed Scopus (230) Google Scholar), and it also acts as a MAPK kinase kinase (44Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar) whose involvement in regulation of maturation is subject to controversy. Using a dominant negative mutant approach it has been reported (45Muslin A.J. Mac Nicol A. Williams L.T. Mol. Cell. Biol. 1993; 13: 4197-4202Crossref PubMed Scopus (98) Google Scholar) that Raf-1 activation is required for progesterone-induced maturation, but this result was challenged (46Fabian J.R. Morrison D.K. Daar I. J. Cell Biol. 1993; 122: 645-652Crossref PubMed Scopus (119) Google Scholar). It was recently shown that autophosphorylation of Raf-1 on serine 621 (47Mischak H. Seitz T. Janosch P.K. Enlitz M. Steen H. Schellerer M. Philipp A. Kolch W. Mol. Cell. Biol. 1996; 16: 5409-5418Crossref PubMed Scopus (178) Google Scholar) is a way to down-regulate its activity, and this same site can also be phosphorylated by PKA. We discussed in a previous paper a possible transient activation of Raf-1 in Xenopus oocytes, upon down-regulation of PKA, during normal maturation induced by progesterone (14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar). Because Raf-1 mobility shift observed during maturation by progesterone is usually equated to the kinase activation, we verified that Raf-1 is indeed prevented from shifting upon progesterone treatment of oocytes injected with X-PAK1-Cter (data not shown). As X-PAK1 belongs to the well known family of PAK proteins that were first isolated as upstream regulators of MAPK cascades, involvement of X-PAK in Raf-1 kinase regulation is an interesting hypothesis.We show that X-PAK1-Cter negatively targets the same events in inhibition of maturation as PKA/C and moreover that X-PAK1-Cter prevents maturation induced by the regulatory subunit of protein kinase A, but we have been unable to demonstrate any direct activation of endogenous X-PAK1 or two other closely related family members X-PAK2 and X-PAK3 by PKA/C. Both pathways could also be linked in a different manner; for example, X-PAK could interfere with the ability of PKA to form a tetramer (for example by phosphorylation of PKA/R) and consequently inhibit its inactivation. Indeed it has previously been demonstrated that PKA/RII can be phosphorylated in vitro by different kinases, and recently Keryer et al. (41Keryer G. Yassenko M. Labbé J.C. Castro A. Lohmann S.M. Evain-Brion D. Taskén K. J. Biol. Chem. 1998; 273: 34594-34602Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) reported that MPF can also phosphorylate it. We verified that neither PKA/R nor PKA/C are substrates of X-PAK1-Cter. At the moment we cannot rule out other possible cross-links in the pathways used by the two kinases; however, another possible explanation of our results would be that both pathways are parallel and that active X-PAK and PKA target an identical substrate that allows the MPF amplification loop to be turned on.Both PKA and X-PAK kinases are also capable of inhibiting the G2/M transition during the first mitotic cycle, at which point cyclin B-p34cdc2 is still regulated by phosphorylation on Tyr15, but control by MAPK activity is absent. We show that X-PAK1-Cter does not prevent cyclin A-p34cdc2 activation in cell-free egg cycling extracts, and in fact the kinase reaches an abnormally high level of activity compared with a control cycling extract supplemented with MalBP fusion protein. Nevertheless, under these conditions, high cyclin A-p34cdc2activity does not allow cyclin B-p34cdc2 activation, and as a consequence the cyclin degradation pathway is not turned on. We show that cyclin B-p34cdc2 complexes are indeed formed, but no H1 activity is associated with them, and the p34cdc2 subunit displays a slow electrophoretic mobility indicative of phosphorylation on Tyr15. We conclude that X-PAK1-Cter activity is very specific because it does not interfere with the activity of cyclin A-p34cdc2 complexes, which are not subject to a regulation by phosphorylation on Tyr15 (30Abrieu A. Brassac T. Galas S. Fisher D. Labbé J.C. Dorée M. J. Cell Sci. 1998; 111: 1751-1757Crossref PubMed Google Scholar, 48Clarke P.R. Leiss D. Pagano M. Karsenti E. EMBO J. 1992; 11: 1751-1761Crossref PubMed Scopus (61) Google Scholar), but it does interfere specifically with the activation of cyclin B-p34cdc2 complexes by preventing dephosphorylation of Tyr15. In 1994 Griecoet al. (49Grieco D. Avvedimento E. Gottesman M.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9896-9900Crossref PubMed Scopus (51) Google Scholar) showed that PKA activity oscillates during the first embryonic cycles in Xenopus extracts, being high at interphase and falling at the onset of mitosis. They demonstrated that sustained PKA activity inhibits cycle progression in a dose-dependent manner and that extracts eventually arrest in interphase with a high level of inactive pre-MPF complexes. In this context, X-PAK1–1Cter and PKA probably act independently of a control over MAPK activity, because MAPK is inactive in the cycling extract, and so this cannot be compared with the G2/M transition block described previously (50Abrieu A. Fisher D. Simon M.N. Dorée M. Picard A. EMBO J. 1997; 21: 6407-6413Crossref Scopus (72) Google Scholar), in which the proto-oncogene c-Mos is added to cycling extracts. The work of Grieco et al. (49Grieco D. Avvedimento E. Gottesman M.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9896-9900Crossref PubMed Scopus (51) Google Scholar) indicates that PKA could stimulate an okadaic acid-sensitive serine/threonine phosphatase pathway that dephosphorylates Cdc25.Plx kinase is another Cdc25 positive regulatory kinase (36Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar) recently shown (30Abrieu A. Brassac T. Galas S. Fisher D. Labbé J.C. Dorée M. J. Cell Sci. 1998; 111: 1751-1757Crossref PubMed Google Scholar) to be required for the G2/M transition during the first mitotic cell cycle in Xenopus eggs. Cyclin B-p34cdc2 enhances Plx activation, which triggers Cdc25 and MPF activation in a positive feedback loop. Adding blocking anti-Plx antibodies to an egg cycling extract suppressed entry into mitosis, although a strong cyclin A-p34cdc2 activity is detected, as we observed ourselves in the X-PAK1-Cter block in cycling extracts. Moreover, pre-MPF complexes also remain inactive and phosphorylated on Tyr15, if Plx is blocked with these antibodies. We did not investigate the status of Plx in cycling extracts incubated with X-PAK1-Cter, but we show that during the maturation process Plx activation is prevented by active Xenopus p21-activated kinase.In summary, our data demonstrate that PKA and X-PAK pathways target the same components in their inhibition of G2/M progression during meiosis as well as in the first mitotic cycle inXenopus and that their transduction pathways are likely to be connected at the point where the MPF amplification loop is turned on. We are currently in the process of isolating physiological substrates of X-PAK active kinase in Xenopus extracts to get new insights into factors regulating the G2/M transition. In Xenopus laevis, oocytes are naturally arrested at the G2/M transition of meiosis I. Upon release of progesterone by the follicle cells surrounding the oocytes, maturation occurs and the cell cycle resumes. Translation of the proto-oncogene c-Mos (a MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown.1The abbreviations used are: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PKA, protein kinase A; PAK, p21-activated kinase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Plx, Polo-like kinase; GVBD, germinal vesicle breakdown; NEBD, nuclear envelope breakdown. kinase kinase), is an essential preliminary event (2Sagata N. Oskarsson M. Copeland T. Brumbaugh J. Vande Woude G.F. Nature. 1988; 335: 519-525Crossref PubMed Scopus (462) Google Scholar, 3Kanki J.P. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5794-5798Crossref PubMed Scopus (71) Google Scholar) in turning on the MAPK cascade, which eventually results in almost simultaneous activation of MAPK and maturation-promoting factor (MPF), a heterodimer composed of p34cdc2 kinase and cyclin B (4Labbé J.C. Capony J.P. Caput D. Cavadore J.C. Derancourt J. Kaghad M. Lelias J.M. Picard A. Dorée M. EMBO J. 1989; 8: 3053-3058Crossref PubMed Scopus (375) Google Scholar, 5Gautier J. Maller J.L. EMBO J. 1991; 10: 177-182Crossref PubMed Scopus (155) Google Scholar). Eggs eventually arrest again at metaphase of second meiosis with high MPF and cytostatic factor (in which c-Mos participates (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar)) activities until fertilization. Conversion of inactive pre-MPF complexes, stored in oocytes, into active MPF requires dephosphorylation of residues Thr14 and Tyr15 on p34cdc2 kinase (7Strausfeld U. Labbé J.C. Fesquet D. Cavadore J.C. Picard A. Sadhu K. Russell P. Dorée M. Nature. 1991; 351: 242-245Crossref PubMed Scopus (443) Google Scholar). Dephosphorylation of these amino acids is crucial for MPF activation, and this process is highly regulated by an activating phosphatase, Cdc25 (for review see Ref. 8Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar), and two inhibitory kinases, Myt1 (9Mueller P.R. Coleman T.R. Kumagai A. Dunphy W.G. Science. 1995; 270: 86-90Crossref PubMed Scopus (533) Google Scholar) and Wee1 (10Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (268) Google Scholar). Although oocyte maturation has been extensively studied over the past 10 years as a model to understand the mechanisms involved in reentry into the cell cycle, the signal transduction pathway between progesterone binding to its receptor and the activation of the MAPK cascade and MPF is ill understood. Protein kinase A (PKA) appears to be a crucial player in these events, because activation of progesterone receptor is followed by a sudden drop in cAMP concentration (11Speaker M.C. Butcher F.R. Nature. 1977; 267: 848-849Crossref PubMed Scopus (105) Google Scholar) and likely a subsequent inactivation of protein kinase A. Indeed, microinjection of the PKA catalytic subunit prevents progesterone-induced maturation in oocytes, whereas the expression of the PKA regulatory subunit is sufficient to induce maturation (12Maller J. Krebs E. J. Biol. Chem. 1977; 252: 1712-1718Abstract Full Text PDF PubMed Google Scholar). Matten et al. (13Matten W. Daar I. Vande Woude G.F. Mol. Cell. Biol. 1994; 14: 4419-4426Crossref PubMed Scopus (109) Google Scholar) reported that PKA negatively regulates maturation by controlling both c-Mos translation and Cdc25 activation. How PKA acts on c-Mos de novo translation is, however, unclear, and we have demonstrated that in conditions in oocytes in which activation of MAPK is allowed, PKA does not inhibit the c-Mos translation (14Faure S. Morin N. Dorée M. Oncogene. 1998; 17: 1215-1221Crossref PubMed Scopus (20) Google Scholar), raising the possibility that PKA could act on a single target in the negative regulation of maturation. A number of studies demonstrate the importance of the MAPK cascade in MPF activation (6Sagata N. Watanabe N. Vande Woude G.F. Ikawa Y. Nature. 1989; 342: 512-518Crossref PubMed Scopus (524) Google Scholar, 15Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 9: 2131-2138Crossref Scopus (189) Google Scholar) during Xenopus oocyte maturation. Indeed, microinjection of recombinant c-Mos is itself sufficient to induce oocyte maturation in the absence of hormonal treatment (16Yew N. Mellini M.L. Vande Woude G.F. Nature. 1992; 355: 649-652Crossref PubMed Scopus (202) Google Scholar, 17Sagata N. Daar I. Oskarsson M. Showalter S.D. Vande Woude G.F. Science. 1989; 245: 643-646Crossref PubMed Scopus (249) Google Scholar), whereas CL100 (MAPK phosphatase-1) inhibits progesterone-induced maturation (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Important work in Saccharomyces cerevisiaetoward the understanding of the control of the MAPK cascade has been undertaken in the last few years. The response to the mating pheromone signal is mediated by the direct interaction of the Gβ subunit of G protein heterotrimers to Ste20, a serine threonine kinase (19Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-194Crossref PubMed Scopus (183) Google Scholar). Activation of Ste20 results in activation of the MAPK kinase kinase Ste11, which allows the MAPK cascade to be turned on and results in MAPK (Kss1 and Fus3) activation (reviewed in Ref. 20Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. Dev. 1997; 7: 162-167Crossref Scopus (188) Google Scholar). InXenopus oocytes, Gotoh et al. (18Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) showed that microinjection of constitutively active Ste11 is able to efficiently induce the maturation process. Moreover, addition of the N-terminal regulatory domain of Ste11 to Xenopus extracts prevents a constitutive form of Ste20 from activating MAPK (21Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). p21-activated kinases (PAKs) are Ste20 homologues. Understanding of their functional importance in signal transduction and in regulation of the actin cytoskeleton is rapidly growing (see Refs. 22Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 23Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar, 24Manser E.H.Y. Huang T.H. Loo X.Q. Chen J.M. Dong T. Leung L. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar). We have cloned Xenopus PAKs and recently demonstrated that a member of this family is involved in regulation of the oocyte maturation process (1Faure S. Vigneron S. Dorée M. Morin N. EMBO J. 1997; 16: 5550-5561Crossref PubMed Scopus (73) Google Scholar). Indeed, we showed that microinjection of a constitutive truncated version of X-PAK1 (X-PAK1-Cter) into oocytes completely blocks both insulin and progesterone mediated maturation, and converse
Greatwall (GWL) is an essential kinase that indirectly controls PP2A-B55, the phosphatase counterbalancing cyclin B/CDK1 activity during mitosis. In Xenopus laevis egg extracts, GWL-mediated phosphorylation of overexpressed ARPP19 and ENSA turns them into potent PP2A-B55 inhibitors. It has been shown that the GWL/ENSA/PP2A-B55 axis contributes to the control of DNA replication, but little is known about the role of ARPP19 in cell division. By using conditional knockout mouse models, we investigated the specific roles of ARPP19 and ENSA in cell division. We found that Arpp19, but not Ensa, is essential for mouse embryogenesis. Moreover, Arpp19 ablation dramatically decreased mouse embryonic fibroblast (MEF) viability by perturbing the temporal pattern of protein dephosphorylation during mitotic progression, possibly by a drop of PP2A-B55 activity inhibition. We show that these alterations are not prevented by ENSA, which is still expressed in Arpp19Δ/Δ MEFs, suggesting that ARPP19 is essential for mitotic division. Strikingly, we demonstrate that unlike ARPP19, ENSA is not required for early embryonic development. Arpp19 knockout did not perturb the S phase, unlike Ensa gene ablation. We conclude that, during mouse embryogenesis, the Arpp19 and Ensa paralog genes display specific functions by differentially controlling cell cycle progression.
Here we investigate the mechanisms regulating Greatwall (Gwl), a serine/threonine kinase essential for promoting the correct timing of mitosis. We identify Gwl as a unique AGC kinase that, unlike most AGC members, appears to be devoid of a hydrophobic motif despite the presence of a functional hydrophobic pocket. Our results suggest that Gwl activation could be mediated by the binding of its hydrophobic pocket to the hydrophobic motif of another AGC kinase. Our molecular modeling and mutagenic analysis also indicate that Gwl displays a conserved tail/linker site whose phosphorylation mediates kinase activation by promoting the interaction of this phosphorylated residue with two lysines at the N terminus. This interaction could stabilize the αC-helix and maintain kinase activity. Finally, the different phosphorylation sites on Gwl are identified, and the role of each one in the regulation of Gwl kinase activity is determined. Our data suggest that only the phosphorylation of the tail/linker site, located outside the putative T loop, appears to be essential for Gwl activation. In summary, our results identify Gwl as a member of the AGC family of kinases that appears to be regulated by unique mechanisms and that differs from the other members of this family.
