Cloning and Characterization of Xenopus Rsk2, the Predominant p90 Rsk Isozyme in Oocytes and Eggs

Abstract

The 90-kDa ribosomal S6 kinases, the p90 Rsks, are a family of intracellular serine/threonine protein kinases distinguished by two distinct kinase domains. Rsks are activated downstream of the ERK1 (p44) and ERK2 (p42) mitogen-activated protein (MAP) kinases in diverse biological contexts, including progression through meiotic and mitotic M phases in Xenopus oocytes and cycling Xenopus egg extracts, and are critical for the M phase functions of Xenopus p42 MAPK. Here we report the cloning and biochemical characterization of Xenopus Rsk2. Xenopus Rsk1 and Rsk2 are specifically recognized by commercially available RSK1 and RSK2 antisera on immunoblots, but both Rsk1 and Rsk2 are immunoprecipitated by RSK1, RSK2, and RSK3 sera. Rsk2 is about 20-fold more abundant than the previously described Xenopus Rsk1 protein; their concentrations are approximately 120 and 5 nM, respectively. Rsk2, like Rsk1, forms a heteromeric complex with p42 MAP kinase. This interaction depends on sequences at the extreme C terminus of Rsk2 and can be disrupted by a synthetic peptide derived from the C-terminal 20 amino acids of Rsk2. Finally, we demonstrate that p42 MAP kinase can activate recombinant Rsk2 in vitro to a specific activity comparable to that found in Rsk2 that has been activated maximally in vivo. These findings underscore the importance of the Rsk2 isozyme in the M phase functions of p42 MAP kinase and provide tools for further examining Rsk2 function.The mitogen-activated protein kinase (MAPK) 1 cascade is a three-kinase module comprising a MAPK, a MAP kinase kinase (MAPKK), and a MAP kinase kinase kinase (MAPKKK) (1-3).One of the best-studied MAPK cascades consists of the p42 and p44 (or ERK2 and ERK1) MAPKs, the MEK1 and MEK2 MAPKKs, and the Raf and Mos MAPKKKs. Raf and Mos can phosphorylate MEK1 and MEK2 on two serine residues in MEK's T-loop, thereby activating the MEK. Active MEK in turn can phosphorylate p42 or p44 at threonine and tyrosine residues within the sequence Thr-Glu-Tyr in MAPK's T-loop, thereby activating the MAPK.p42 and p44 MAPK are probably best-known for their roles in mitogenesis and cell fate induction (4). However, these proteins have also been implicated in the regulation of meiotic and mitotic M phase. Much of the work on the M phase roles of MAPKs has been carried out in Xenopus laevis oocytes, eggs, and cycling egg extracts, where the relevant MAPK is p42 (ERK2) MAPK (5-8).The first indication that p42 MAPK might play a role in M phase regulation came from studies of Xenopus oocyte maturation. Maturation is a key step in the production of a fertilizable egg, and its study has a long history of providing important insights into the biochemistry of M phase regulation. Fully grown Xenopus oocytes are arrested in a G 2 -like state with an intact germinal vesicle (nucleus) and inactive Cdc2/cyclin B and p42 MAPK. Progesterone releases oocytes from this arrest state and brings about a resumption of meiosis I. Progesteronetreated oocytes undergo germinal vesicle breakdown, complete the first meiotic d...

RSK2 is a kinetochore-associated protein that participates in the spindle assembly checkpoint

“…According to the data reported by other laboratories (Bhatt and Ferrell, 2000;Nishiyama et al, 2007), our data show that Rsk2 depletion does not promote metaphase II exit. Rsk2-depleted CSF extracts presented phosphorylated cyclin B-Cdk1 substrates and condensed Figure 1 Rsk2 localizes at the kinetochores under spindle checkpoint conditions.…”

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

“…M-phase entry is controlled in part by Cdc2, which is a CDK normally kept in check by dual phosphorylation on both Thr14 and Tyr15 by the inhibitory kinase Myt1. RSK2 is the prominent RSK isoform in Xenopus oocytes (24), and using this model system, RSK2 was shown to contribute to the control of the meiotic cell cycle at several critical points (316). One mechanism by which RSK2 participates in the progression of oocytes through the G 2 /M phase of meiosis I is through phosphorylation and inhibition of the Myt1 kinase (137,260,305).…”

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

“…on May 11, 2018 by guest http://mmbr.asm.org/ (8), and with this model system, RSK2 has been shown to contribute to the control of the meiotic cell cycle at several critical points (174). One mechanism by which RSK2 participates in the progression of oocytes through the G 2 /M phase of meiosis I is through phosphorylation and inhibition of the Myt1 kinase (144).…”