title: An analysis of the substrate specificity of insulin-stimulated protein kinase-1, a mammalian homologue of S6 kinase-II.
abstract: The specificity determinants for insulin-stimulated protein kinase-I (ISPK-1) have been investigated with synthetic peptides based on naturally-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 residue further away from the serine, the nonapeptide (Arg)4-Ala-Ala-Ser-Val-Ala being the best substrate among all the short peptides tested (Km = 15 microM). Conversely, the substitution of either Thr for Ser or Lys for Arg is detrimental. Likewise, two flanking Pro residues and an Arg immediately N-terminal to the Ser act as negative specificity determinants. 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 respects including (1), the detrimental effect of a Lys for Arg substitution which is still compatible with some phosphorylation by ISPK-1, but 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 effect of substituting Phe for Val in the peptide Arg-Arg-Ala-Ser-Val-Ala, which improves the efficiency of phosphorylation by PK-A (lowering the Km 4-fold), but has no effect on phosphorylation by ISPK-1. These differences in peptide substrate specificity may account in part for the different rates of phosphorylation of physiological substrates for ISPK-1 and PK-A, such as the G subunit of protein phosphatase-1.
authors.affiliation: Dipartimento di Chimica Biologica, C.N.R., Università di Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Donella-Deana
authors.forename: A
authors.initials: A
authors.lastname: Lavoinne
authors.forename: O
authors.initials: O
authors.lastname: Marin
authors.forename: L A
authors.initials: LA
authors.lastname: Pinna
authors.forename: P
authors.initials: P
authors.lastname: Cohen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007328:Insulin / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D038601:Ribosomal Protein S6; D019893:Ribosomal Protein S6 Kinases; D012269:Ribosomal Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases