Evidence that a novel serine kinase catalyses phosphorylation of the insulin receptor in an insulin-dependent and tyrosine kinase-dependent manner

Abstract

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this 'in vitro' system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2" only), treatment at 37°C (1 h), N-ethylmaleimide, phosphate, /3-glycerol phosphate and antiphosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2", Ca2++calmodulin, Ca2++phosphatidylserine+diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2". This indicates that it is distinct from Ca2 , Ca2 /phospholipid, Ca2 /calmodulin, cyclic AMP-and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase. INTRODUCTIONThe insulin receptor is an insulin-activated tyrosinespecific protein kinase which catalyses the autophos-

Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain

“…These data are in agreement with previous reports showing that a synthetic peptide corresponding to the major autophosphorylation domain of the insulin receptor is a good phosphorylation substrate and potent kinase inhibitor [21][22][23][24][25][26][27]. As suggested by Shoelson et al [24], kinase inhibition by this peptide occurs by a complex mechanism that probably involves inhibition of receptor autophosphorylation (which in turn inhibits kinase activation) and competition for phosphorylation of exogenous substrates.…”

Identification of Ser1275 and Ser1309 as Autophosphorylation Sites of the Human Insulin Receptor in Intact Cells

“…Phorbol ester and forskolin are known to stimulate serine/threonine phosphorylation of the insulin receptor (21)(22)(23), presumably by activation of isoforms of protein kinase C (PKC) and cAMP dependent protein kinase (PKA), respectively (24 -26). Furthermore, the ␤-subunit of the IR is phosphorylated by an insulinstimulated serine kinase, which has been postulated to be associated with the receptor (16,19,26,27). We (17, 28 -30) and others (18,19,31) on the other hand, provided strong evidence that the IR contains intrinsic serine/threonine-as well as tyrosine-kinase activity suggesting "dual specificity" (32) of the kinase domain.…”

Activation of phosphatidylinositol 3-kinase by insulin.

“…It is possible that a product of the PtdIns 3-kinase mediates one or more of these responses. Recent findings in this laboratory (unpublished data) and others (28)(29)(30)(31) (36).…”