The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation

Huchon, Denise; Ozon, René; Fischer, Edmond H.; Demaille, Jacques

doi:10.1016/0303-7207(81)90092-7

Cited by 92 publications

(50 citation statements)

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“…It has long been known that PKA activity is required to maintain oocytes of all species tested in their natural G 2 arrest and that a drop in PKA activity is required for the G 2 /meiosis I transition (O'Connor and Smith, 1976;Maller and Krebs, 1977;Speaker and Butcher, 1977;Maller et al, 1979;Mulner et al, 1979;Finidori-Lepicard et al, 1981;Huchon et al, 1981;Sadler and Maller, 1981;Masui, 1985). Phosphorylation of cdc25C Ser287 by PKA has recently been shown to be required to maintain the G 2 arrest (Duckworth et al, 2002;Schmitt and Nebreda, 2002).…”

Section: Wee1 and Cdc25 Phosphorylationmentioning

confidence: 99%

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

Stanford

Ruderman

2005

MBoC

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Entry into mitosis is catalyzed by cdc2 kinase. Previous work identified the cdc2-activating phosphatase cdc25C and the cdc2-inhibitory kinase wee1 as targets of the incomplete replication-induced kinase Chk1. Further work led to the model that checkpoint kinases block mitotic entry by inhibiting cdc25C through phosphorylation on Ser287 and activating wee1 through phosphorylation on Ser549. However, almost all conclusions underlying this idea were drawn from work using recombinant proteins. Here, we report that in the early Xenopus egg cell cycles, phosphorylation of endogenous cdc25C Ser287 is normally high during interphase and shows no obvious increase after checkpoint activation. By contrast, endogenous wee1 Ser549 phosphorylation is low during interphase and increases after activation of either the DNA damage or replication checkpoints; this is accompanied by a slight increase in wee1 kinase activity. Blocking mitotic entry by adding the catalytic subunit of PKA also results in increased wee1 Ser549 phosphorylation and maintenance of cdc25C Ser287 phosphorylation. These results argue that in response to checkpoint activation, endogenous wee1 is indeed a critical responder that functions by repressing the cdc2-cdc25C positive feedback loop. Surprisingly, endogenous wee1 Ser549 phosphorylation is highest during mitosis just after the peak of cdc2 activity. Treatments that block inactivation of cdc2 result in further increases in wee1 Ser549 phosphorylation, suggesting a previously unsuspected role for wee1 in mitosis. INTRODUCTIONEntry into mitosis is initiated by activation of cyclin B/cdc2. Preformed complexes of cyclin B/cdc2 accumulate during interphase, but their activity is repressed by inhibitory phosphorylations on cdc2 at Tyr15 (catalyzed by wee1 and myt1) and Thr14 (catalyzed by myt1). These phosphorylations are removed by the phosphatase cdc25C (reviewed in Berry and Gould, 1996;Lew and Kornbluth, 1996). Early work led to the conclusion that cdc2 and cdc25C activities both increase rapidly during the G 2 /M transition as the result of positive feedback loops between cyclin B/cdc2 and cdc25C, ultimately leading to the full activation of both cdc25C and cyclin B/cdc2 (Izumi et al., 1992;Hoffmann et al., 1993;Izumi and Maller, 1993;Strausfeld et al., 1994). The mechanisms that trigger this abrupt activation are still not well understood (Margolis and Kornbluth, 2004;Perdiguero and Nebreda, 2004).Over the past decade, there has been a large increase in the molecular understanding of how checkpoint pathways become activated, and how they affect the basic cell cycle regulatory machinery. Genetic and biochemical approaches have identified sensors that detect incompletely replicated or damaged DNA and that stimulate signal transduction pathways that lead to activation of the kinases Chk1 and Chk2/Cds1 (reviewed in Sancar et al., 2004). Both Chk1 and Chk2 can phosphorylate cdc25C on Ser287 (human Ser216), which creates a binding site for 14-3-3 proteins; this is thought to inhibit cdc25C activation and thus...

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Section: Wee1 and Cdc25 Phosphorylationmentioning

confidence: 99%

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

Stanford

Ruderman

2005

MBoC

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“…It has long been known that protein kinase A (PKA) negatively regulates oocyte activation in vertebrates and most other species (11)(12)(13)(14)(15)(16)(17)(18)(19). In Xenopus oocytes, progesterone induces a sudden drop in adenylyl cyclase activity and cAMP.…”

mentioning

confidence: 99%

G ₂ arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

Duckworth

Weaver

Ruderman

2002

Proc. Natl. Acad. Sci. U.S.A.

196

209

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Xenopus oocytes, which are arrested in G2 of meiosis I, contain complexes of cyclin B-cdc2 (M phase-promoting factor) that are kept repressed by inhibitory phosphorylations on cdc2 at Thr-14 and Tyr-15. Progesterone induces a cytoplasmic signaling pathway that leads to activation of cdc25, the phosphatase that removes these phosphorylations, catalyzing entry into M phase. It has been known for 25 years that high levels of cAMP and protein kinase A (PKA) are required to maintain the G 2 arrest and that a drop in PKA activity is required for M phase-promoting factor activation, but no physiological targets of PKA have been identified. We present evidence that cdc25 is a critical target of PKA. (i) In vitro, cdc25 Ser-287 serves as a major site of phosphorylation by PKA, resulting in sequestration by 14-3-3. (ii) Endogenous cdc25 is phosphorylated on Ser-287 in oocytes and dephosphorylated in response to progesterone just before cdc2 dephosphorylation and M-phase entry. (iii) High PKA activity maintains phosphorylation of Ser-287 in vivo, whereas inhibition of PKA by its heat-stable inhibitor (PKI) induces dephosphorylation of Ser-287. (iv) Overexpression of mutant cdc25 (S287A) bypasses the ability of PKA to maintain oocytes in G 2 arrest. These findings argue that cdc25 is a physiologically relevant target of PKA in oocytes. In the early embryonic cell cycles, Ser-287 is phosphorylated during interphase and dephosphorylated just before cdc2 activation and mitotic entry. Thus, in addition to its role in checkpoint arrest, cdc25 Ser-287 serves as a site for regulation during normal, unperturbed cell cycles.

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“…It is interesting to note that recent studies on NEM treatment of PKAc show that Cys-199 is the preferred target for NEM and that alkylation at this site impairs not only catalytic activity but also the ability of PKAc to interact with both R subunits. 4 In light of the results presented here, it may be that the failure to inhibit GVBD by NEM-treated PKAc was due to the failure to interact with R subunits, rather than to the loss of catalytic activity by alkylation.…”

Section: Fig 4 Effect Of Regulatory Subunit Interaction On Inhibitimentioning

confidence: 74%

“…Overexpression of a non-physiological PKAc substrate in oocytes has confirmed predictions of a decrease in PKAc activity with progesterone treatment (34); however, kinase assays with crude extracts and similar nonspecific substrates have not revealed a detectable increase in total extract PKAc activity after expression of K72R 3 T. Hunter, personal communication. 4 S. Taylor, personal communication.…”

Section: Fig 4 Effect Of Regulatory Subunit Interaction On Inhibitimentioning

confidence: 99%

“…The dependence of G 2 maintenance on the second messenger cAMP was first discovered in amphibian oocytes, which decrease cAMP in response to progesterone to release the G 2 arrest and re-initiate the meiotic cell cycle (1,2). The enormous size of these oocytes and the resulting ability to microinject them facilitated early experiments that showed that injection of the catalytic subunit of the cAMP-dependent protein kinase (PKAc) 1 blocked the ability of progesterone to initiate oocyte maturation and that microinjection of either the regulatory (R) subunit of PKA (of which there are two termed RI and RII) or the heat-stable inhibitor of PKA, PKI, led to re-initiation of oocyte maturation in the absence of progesterone (3,4). The demonstration that a decrease in cAMP and PKAc was both necessary and sufficient for meiosis re-initiation was perhaps the first demonstration that all of the effects of a cAMP change could be explained by changes in the activity of PKA, although other targets of cAMP have since been identified (5,6).…”

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confidence: 99%

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Regulation of the G2/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase

Eyers

Liu

Hayashi

et al. 2005

Journal of Biological Chemistry

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Vertebrate oocytes are arrested in G 2 phase of the cell cycle at the prophase border of meiosis I. Progesterone treatment of Xenopus oocytes releases the G 2 block and promotes entry into the M phases of meiosis I and II. Substantial evidence indicates that the release of the G 2 arrest requires a decrease in cAMP and reduced activity of the cAMP-dependent protein kinase (PKAc). It has been reported and we confirm here that microinjection of either wild type or kinase-dead K72R PKAc inhibits progesterone-dependent release of the G 2 arrest with equal potency and that inhibition can be reversed by a second injection of the heat-stable inhibitor of PKAc, PKI. However, a mutant enzyme predicted to be completely kinase-dead from the crystal structure of PKAc, K72H PKAc, was much less inhibitory when carrying additional mutations that block interaction with either type I or type II regulatory subunit. Moreover, inhibition by K72H PKAc was reversed by PKI at a 30-fold lower concentration and with more rapid kinetics compared with wild type PKAc. K72R PKAc was found to have low but detectable activity after incubation in an oocyte extract. These results indicate that inhibition of the progesterone-dependent G 2 /M transition in oocytes after microinjection of dead PKAc reflects either low residual activity or binding to regulatory subunits with a resulting net increase in the level of endogenous wild type PKAc. Consistent with this hypothesis, the induction of mitosis in Xenopus egg extracts by the addition of cyclin B was blocked by wild type PKAc but not by K72H PKAc. The identification of substrates for PKAc that maintain cell cycle arrest in G 2 remains an important goal for future work.

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The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation

Cited by 92 publications

References 21 publications

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

G ₂ arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

Regulation of the G2/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase

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The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation

Cited by 92 publications

References 21 publications

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

G 2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A

Regulation of the G2/M Transition in Xenopus Oocytes by the cAMP-dependent Protein Kinase

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G ₂ arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A