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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 p42MAPK. 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 p42MAP 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 p42MAP 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 p42MAP kinase and provide tools for further examining Rsk2 function.
Figure 1
The cDNA sequence of XenopusRsk2. A, the predicted amino acid sequence of theXenopus Rsk2, human RSK2, human RSK3, XenopusRsk1α, and human RSK1. Sequences were aligned using ClustalW 1.8 on the Baylor College of Medicine multiple sequence alignment server on the Web. Minor adjustments to the alignment were made manually. The highlighted residues (white letters on black backgrounds) are those that are conserved betweenXenopus Rsk2 and one or more of the other Rsk sequences. Thegray boxes denote the N-terminal and C-terminal kinase domains. The circled asterisks denote the phosphorylation sites identified by Dalby et al. (31). B, percentage identities between Xenopus Rsk2 and various other Rsk isozymes.
Figure 2
Phylogram trees for the Rsk N-terminal and C-terminal kinase domains. Neighbor-joining trees were generated from the kinase domain alignments shown in Fig. 1 by means of the Macintosh version of ClustalX. The N-terminal and C-terminal kinase domains are plotted on the same scale. The scale barrepresents 0.1 differences per site.
Figure 3
Immunoblots of Xenopus Rsk1 and Rsk2. Samples were prepared from G2 phase and M phase oocytes, blotted, and probed with human RSK1 (left) or RSK2 (right) antisera. The molecular masses shown on the right were taken from prestained molecular weight markers.
Figure 4
The abundance of Rsk1 and Rsk2 in oocytes. Recombinant Î43-His6-Rsk1 and His6-Rsk2 were purified and used as standards for Rsk1 and Rsk2 blots. A, Rsk1 blot of Î43-His6-Rsk1 standards and lysates equivalent to four oocytes per lane.B, quantitative data from phosphorimaging analysis of the blot shown in A. C, Rsk2 blot of His6-Rsk2 standards and lysates equivalent to four oocytes per lane. D, quantitative data from densitometry of the blot shown in C.
Figure 5
Immunoprecipitation of Rsk1 and Rsk2 by various RSK antisera. A, RSK1, RSK2, and RSK3 sera bring down similar amounts of Rsk1 and Rsk2. Lysates from G2 phase Xenopus oocytes were subjected to immunoprecipitation using RSK1, RSK2, or RSK3 antiserum, or protein G beads alone. The immunoprecipitates were subjected to 10.5% SDS-PAGE, followed by immunoblot analysis, as indicated above. The 68-kDa prestained molecular mass marker was run until 1 cm from bottom of gel to enhance separation of Rsk1 from Rsk2. B, kinase activities of RSK immunoprecipitates. Lysates from G2- or M phase oocytes were subjected to immunoprecipitation using RSK1, RSK2, or RSK3 antiserum, or protein G beads alone. The immunoprecipitates were washed and subjected to S6 kinase assay with an S6 peptide substrate and [γ-32P]ATP. Results were quantified by Cerenkov counting.
Figure 6
Complex formation between Rsk2 and p42 MAPK. A, p42 MAPK is pulled down with Rsk2. Lysates from Sf9 cells expressing either His6-Rsk2 or GST·MAPK were combined and incubated at 4â°C for 90 min with protein G-agarose beads and anti-His6 antibodies, both individually and together. His6-Rsk2 and any Rsk2-associated proteins were pulled down with anti-His6antibodies. The immunoprecipitates were then immunoblotted for Rsk2 and p42 MAPK. B, Rsk2 was pulled down with p42 MAPK. Lysates from Sf9 cells expressing His6-Rsk2 or GST·MAPK were combined with glutathione-agarose beads both individually and together and processed as in A. C, binding of endogenous Xenopus p42 MAPK to His6-Rsk2. G2 phase Xenopus oocyte lysates were incubated for 20 min at 4â°C in the presence or absence of purifiedXenopus His6-Rsk2 protein. These lysates were then incubated with protein G-agarose beads in the presence and absence of anti-His6 antibody and incubated for 90 min at 4â°C. The first lane represents the total lysate used in each immunoprecipitation. After centrifugation and washing, the beads were resuspended in sample buffer and resolved by 10.5% SDS-PAGE, followed by p42 MAPK immunoblotting. D, Xenopus egg extracts containing both phosphorylated and unphosphorylated MAPK were treated similarly to C except that lysates were preincubated in the presence or absence of a Rsk2 C-terminal peptide for 10 min at 22â°C prior to adding His6-Rsk2 protein. Beads were collected by centrifugation, washed, and processed as inC.
Figure 7
Activation of Rsk2 in vitro. A, hyperphosphorylation and Ser-383 phosphorylation of His6-Rsk2 in response to MEK R4F and p42 MAPK. Purified recombinant His6-Rsk2 (wild type or K97R mutant) was incubated with constitutively active MEK R4F with or without p42 MAPK, as indicated, for 60 min at 30â°C. The reaction products were subjected to SDS-PAGE followed by immunoblotting with RSK2 antibodies (top blot) or phospho-Rsk antibodies (bottom blot) that detected Rsk2 when it was phosphorylated at Ser-383. B, activation of His6-Rsk2 by MEK R4F and p42 MAPK. His6-Rsk2 was incubated as described inA and tested for S6 kinase activity as described in Fig.5.
