Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

• DOI: 10.1128/mcb.11.11.5541
• Publication Date: 2015-10-06T00:27:58Z
• Authors: J R Grove; P Banerjee; A Balasubramanyam; P J Coffer; D J Price; J Avruch; J R Woodgett
• Keywords: 0301 basic medicine; Base Sequence; Transcription, Genetic; Ribosomal Protein S6 Kinases; Molecular Sequence Data; Gene Expression; Transfection; Cell Line; Rats; Isoenzymes; Molecular Weight; 03 medical and health sciences; Liver; Protein Biosynthesis; Sequence Homology, Nucleic Acid; Animals; Humans; Amino Acid Sequence; Cloning, Molecular; Protein Kinases; Gene Library

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