CLONING AND EXPRESSION OF 2 HUMAN P70 S6 KINASE POLYPEPTIDES DIFFERING ONLY AT THEIR AMINO TERMINI

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha-I; calculated M(r) 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptide encoded by the second cDNA (p70 S6 kinase alpha-II; calculated M(r) 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha-I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha-I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent M(r) of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha-I and alpha-II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha-II) and 85 and 90 kDa (alpha-I). Transfection with the alpha-II cDNA yields only the smaller set of bands, while transfection with the alpha-I cDNA generates both sets of bands. Mutation of Met-24 in the alpha-I cDNA to Leu or Thr suppresses synthesis of the alpha-II polypeptides. Only the p70 alpha-I and alpha-II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70-and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.