Direct roles of the signaling kinase RSK2 in Cdc25C activation during Xenopus oocyte maturation

Abstract

The induction of M phase in eukaryotic cell cycles requires robust activation of Cdc2/cyclin B by Cdc25, which itself is robustly activated by serine/threonine phosphorylations. Although multiple protein kinases that directly activate Cdc25C have been identified, whether the combination of different primary phosphorylations of Cdc25C is sufficient to fully activate Cdc25C has not been determined. By analyzing the GST-Cdc25C phosphorylating activity in Xenopus egg extracts, we previously defined roles of MAPK and Cdc2/cyclin B in partially activating Cdc25C and predicted the presence of another major Cdc25C-activating kinase. In this study, we demonstrate that this missing kinase is RSK2, which phosphorylates three sites in Cdc25C and also partially activates Cdc25C. However, the phosphorylations catalyzed by MAPK, Cdc2, and RSK2 fail to fully activate Cdc25C, suggesting that additional biochemical events are required to fully activate this key cell cycle regulator. by the protein phosphatase Cdc25 (1, 2). At onset of M phase, Cdc25C activity increases 10-to 20-fold due to extensive phosphorylation of its N-terminal regulatory domain, and the phosphorylation increases the apparent molecular weight of Cdc25C by 20-40 kDa (3-5). In the widely accepted model for Cdc25C activation, a non-Cdc2 kinase (such as a polo-like kinase) is activated before Cdc2 activation, contributing to the initiation of Cdc25C activation. The catalytic amount of activated Cdc25C then activates a small amount of Cdc2/cyclin B, which in turn activates more Cdc25C and vice versa. This direct positive feedback loop produces concurrent and switch-like activation of Cdc2/ cyclin B and the dramatic gel mobility shift of Cdc25C, indicative of Cdc25C full activation (6, 7).Although the above model is consistent with the "all or none" characteristics of M-phase induction and the autocatalytic nature of maturation-promoting factor (MPF) and Cdc2/cyclin B in Xenopus oocytes (7-9), several studies have suggested that Cdc25C activation is much more complicated. For example, addition of recombinant Cdc25C to immature Xenopus oocyte extracts induced activation of endogenous Cdc2 and Plx1 but failed to induce the dramatic gel mobility shift of Cdc25C. The latter could, however, be induced if the phosphatase inhibitor okadaic acid (OA) was also added to oocyte extracts (10). In interphase-arrested Xenopus egg extracts depleted of cyclin B, the large mobility shift of Cdc25C could be induced by the phosphatase inhibitor microcystin plus the classical MPF extraction buffer (11) even in the absence of Cdc2 and Cdk2 proteins (12). These results suggest a model whereby full activation of Cdc25C involves mechanistically distinct phosphorylation steps.To understand the complex process of Cdc25C activation during G2/M transition, our strategy has been to identify all major kinases in M phase-arrested Xenopus egg extracts (MEE) that phosphorylate and activate GST-tagged Xenopus Cdc25C (GSTCdc25C). As GST-Cdc25C is unphosphorylated, we term the phosphorylations catal...

RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B

“…Among the numerous potential RSK phosphorylation sites in xCdc25C, RSK2 predominantly phosphorylates S317, T318 and/or S319 in the motif 313 KRRRSTS 319 (36). Pairwise alignment of hCdc25A, hCdc25B and hCdc25C with xCdc25C demonstrated that RSK2 phosphorylation sites in xCdc25C localize in a conserved region near the catalytic domain (Fig.…”

“…Our previous results demonstrated that ERK preferentially phosphorylates and activates hCdc25C in mitotic cells (32). As ERK activation does not depend on the activation of mitotic Cdk, we hypothesize that ERK is one of the initiating kinases for hCdc25C activation during G2/M transition (36). Different from ERK, the results in this study show that RSK preferentially activates hCdc25A and hCdc25B during G2/M transition.…”

“…The second involves ERK1/2-mediated phosphorylation of Cdc25C (32), the protein phosphatase in Xenopus oocytes that activates the Cdk1/cyclin B complex through removing the inhibitory phosphorylations on Cdk1 (33–35). The third involves activation of Cdc25C by RSK2 (36); RSK2 is the major RSK isoform in Xenopus oocytes (37). Since many of the biochemical regulations regulating meiotic cycles of Xenopus oocytes also occur in mitotic cycles of mammalian cells (38), the three mechanisms described above suggest the possibility that activation of the MAPK cascade positively regulates G2/M transition in mammalian cells through phosphorylation of Cdc25 and Myt 1 by ERK and/or RSK family members.…”

RSK Promotes Prostate Cancer Progression in Bone through ING3, CKAP2, and PTK6-Mediated Cell Survival

“…RSKs phosphorylate many proteins, both cytosolic and nuclear (2). The many effects of RSKs on various proteins may contribute to the observations that RSKs mediate wide-ranging cellular processes, including proliferation (6–8), migration (9), and invasion (1). …”

RSK2 protects mice against TNF-induced bone loss

“…RSK2 is a growth-factor-regulated kinase that was reported to mediate cell cycle progression and cell proliferation in numerous cell types and organisms (Anjum and Blenis, 2008;Cho et al, 2005;David et al, 2005;Eisinger-Mathason et al, 2008;Lin et al, 2008;Romeo and Roux, 2011;Wang et al, 2010). We now report the reduced in vitro growth ability of osteoblast lacking RSK2.…”