A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases.

Abstract

We report the molecular cloning of cDNAs for S6 kinase H (S6K11) mRNAs present in Xenopus ovarian tissue. Two cDNAs were isolated by hybridization to oligonucleotide probes designed to encode tryptic peptides isolated from S6KII. The two cDNAs show 91% sequence similarity to each other. These two cDNAs predict proteins of 733 (S6Klla) and 629 (S6KII) amino acids that show 95% sequence similarity over the 629 amino acids where they are colinear. Amino acids 44-733 of S6KIIa were expressed in Escherichia coli and the recombinant protein was used to raise antiserum in rabbits. This antiserum reacted with authentic S6KII prepared from Xenopus eggs. This interaction was specifically blocked by the recombinant protein from E. coli. The sequences of S6KIIa and -13 predict four tryptic peptides whose sequences are identical to four peptides isolated from a tryptic digest of S6KII. The S6KII proteins have a very unusual structure when compared with previously studied protein kinases. They contain two apparent kinase domains, each similar to distinct protein kinases. The amino-terminal 366 amino acids show high sequence similarity to the regions of protein kinase C, the catalytic subunit of cAMP-dependent protein kinase, and cGMP-dependent protein kinase that contain the sites for ATP binding and are believed to be the catalytic centers for phosphotransferase activity. The remainder of the S6 kinase molecule shows high sequence similarity to the ATP-binding and presumed catalytic domain ofthe catalytic subunit of phosphorylase b kinase.The activation of a vigorous serine-specific protein kinase and the concomitant phosphorylation of ribosomes is a highly conserved response of animal cells to mitogenic stimulation. Upon mitogenic stimulation, the entire complement of 6 x 106 ribosomes in cultured cells (1) or the 1012 ribosomes in Xenopus oocytes (2) becomes rapidly phosphorylated (3-8). Nearly all of the phosphate is incorporated into a single protein, S6. This event is mediated by a protein kinase activity that can be detected readily by phosphorylation of 40S subunits in vitro. The regulation of this enzyme activity is of interest because of the wide variety of agents that activate the kinase including serum, oncogene products, and phorbol ester in cultured cells (9-11) and insulin and progesterone in Xenopus oocytes (12, 13). Although these agents initially act through different receptors, the pathways apparently converge to activate a single enzyme or a limited number of distinct enzymes.To date, the most highly purified example of an S6 kinase, denoted S6KII, has been obtained from unfertilized Xenopus eggs (14,15). This enzyme has an apparent Mr of 92,000 as determined by NaDodSO4/PAGE. Antiserum raised against purified S6KII is capable of immunoprecipitating S6 kinase activity that can be measured in an immune complex. Studies with this antiserum have revealed that progesterone-or insulin-stimulated oocytes contain an antigenically related enzyme. Furthermore, anti-S6KII antiserum reacts with an S6 kinase a...

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