AN ANALYSIS OF THE SUBSTRATE-SPECIFICITY OF INSULIN-STIMULATED PROTEIN KINASE-1, A MAMMALIAN HOMOLOG OF S6 KINASE-II

The specificity determinants for insulin-stimulated protein kinase-I ( ISPK-1) have been investigated with synthetic peptides based on natura lly-occurring protein phosphoacceptor sequences. Peptides (Arg-Arg-Xaa -Ser-Xaa) that fulfill the consensus sequence for cyclic-AMP-dependent protein kinase (PK-A) are also phosphorylated readily by ISPK-1. The phosphorylation, efficiency is improved by increasing the number of N- terminal arginine residues and by moving the arginyl cluster one resid ue further away from the serine, the nonapeptide (Arg)4-Ala-Ala-Ser-Va l-Ala being the best substrate among all the short peptides tested (K( m) = 15 muM). Conversely, the substitution of either Thr for Ser or Ly s for Arg is detrimental. Likewise, two flanking Pro residues and an A rg immediately N-terminal to the Ser act as negative specificity deter minants. While the specificity of ISPK-1 shows several similarities to that of PK-A, including an absolute requirement for basic residues on the N-terminal side of the target Ser, it differs in several other re spects including (1), the detrimental effect of a Lys for Arg substitu tion which is still compatible with some phosphorylation by ISPK-1, bu t not PK-A; (2), the presence of C-terminal acidic residues which are tolerated very well by ISPK-1, but are detrimental to PK-A; (3), the e ffect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-A la, which improves the efficiency of phosphorylation by PK-A (lowering the K(m) 4-fold), but has no effect on phosphorylation by ISPK-1. The se differences in peptide substrate specificity may account in part fo r the different rates of phosphorylation of physiological substrates f or ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.