The p57 CDKi integrates stress signals into cell-cycle progression to promote cell survival upon stressThe stress-activated protein kinase p38 phosphorylates p57/Kip2, resulting in enhanced CDK2 inhibition and a cell-cycle delay that helps cells to survive under stress.

Cell-cycle progression is regulated by cyclin-dependent kinases (CDKs). CDK1 and CDK2 can be also activated by noncyclin proteins named RINGO/Speedy, which were identified as inducers of the G2/M transition in Xenopus oocytes. However, it is unclear how XRINGO triggers M phase entry in oocytes. We show here that XRINGO-activated CDKs can phosphorylate specific residues in the regulatory domain of Myt1, a Wee1 family kinase that plays a key role in the G2 arrest of oocytes. We have identified three Ser that are major phosphoacceptor sites for CDK/XRINGO but are poorly phosphorylated by CDK/cyclin. Phosphorylation of these Ser inhibits Myt1 activity, whereas their mutation makes Myt1 resistant to inhibition by CDK/XRINGO. Our results demonstrate that XRINGO-activated CDKs have different substrate specificity than the CDK/cyclin complexes. We also describe a mechanism of Myt1 regulation based on site-specific phosphorylation, which is likely to mediate the induction of G2/M transition in oocytes by XRINGO.

Activation of CDK1 is essential for M-phase entry both in mitosis and meiosis. G2-arrested oocytes contain a pool of CDK1/cyclin B complexes that are maintained inactive because of the phosphorylation of CDK1 on Thr14 and Tyr15 by the Wee1 family protein kinase Myt1, whose inhibition suffices to induce meiosis I entry [1-5]. CDK1/XRINGO and p90Rsk can both phosphorylate and downregulate Myt1 activity in vitro [6, 7]. Here we identify five p90Rsk phosphorylation sites on Myt1 that are different from the CDK1/XRINGO sites, and we show how both kinases synergize during oocyte maturation to inhibit Myt1, ensuring meiotic progression. We found that phosphorylation of Myt1 by CDK1/XRINGO early during oocyte maturation not only downregulates Myt1 kinase activity but also facilitates the recruitment of p90Rsk and further phosphorylation of Myt1. Mutation of the five p90Rsk residues to alanine impairs Myt1 hyperphosphorylation during oocyte maturation and makes Myt1 resistant to the inhibition by p90Rsk. Importantly, Myt1 phosphorylated by p90Rsk does not interact with CDK1/cyclin B, ensuring that the inhibitory phosphorylations of CDK1 cannot take place after meiosis I entry and contributing to the all-or-none meiotic response.

Identifying 14-3-3 isoform-specific substrates and functions may be of broad relevance to cell signaling research because of the key role played by this family of proteins in many vital processes. A multitude of ligands have been identified, but the extent to which they are isoform-specific is a matter of debate. Herein we demonstrate, both in vitro and in vivo, a specific, functionally relevant interaction of human 14-3-3␥ with the molecular scaffold KSR1, which is mediated by the C-terminal stretch of 14-3-3␥. Specific binding to 14-3-3␥ protected KSR1 from epidermal growth factor-induced dephosphorylation and impaired its ability to activate ERK2 and facilitate Ras signaling in Xenopus oocytes. Furthermore, RNA interference-mediated inhibition of 14-3-3␥ resulted in the accumulation of KSR1 in the plasma membrane, all in accordance with 14-3-3␥ being the cytosolic anchor that keeps KSR1 inactive. We also provide evidence that KSR1-bound 14-3-3␥ heterodimerized preferentially with selected isoforms and that KSR1 bound monomeric 14-3-3␥. In sum, we have demonstrated ligand discrimination among 14-3-3 isoforms and shed light on molecular mechanisms of 14-3-3 functional specificity and KSR1 regulation.