1428 zyxwvutsrqpon Biochemistry zyxwvut 1987, 26, 1428-1433 Substrate Specificity and Kinetic Mechanism of Human Placental Insulin Receptor /Kinase Duncan H. Walker, Dhandapani Kuppuswamy, Aruna Visvanathan,t and Linda J. Pike* Howard Hughes Medical Institute, Department zyxwvut of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 631 10 Received August 6, 1986; Revised Manuscript Received October 17, 1986 ABSTRACT: The insulin receptor has been shown to be a protein kinase which phosphorylates its substrates on tyrosine residues. To examine the acceptor specificity of affinity-purified insulin receptor/kinase, hydroxyamino acid containing analogues of the synthetic peptide substrate zyxw Arg-Arg-Leu-Ile-Glu-Asp- Ala-Glu-Tyr-Ala-Ala-Arg-Gly were prepared. Substitution of serine, threonine, or D-tyrosine for L-tyrosine completely ablated the acceptor activity of the synthetic peptides. These peptides, along with a phenyl- alanine-containing analogue, did serve as competitive inhibitors of the insulin receptor/kinase with apparent Ki values in the range of 2-4 mM. These data suggest that the insulin receptor/kinase is specific for tyrosine residues in its acceptor substrate and imply that serine phosphate or threonine phosphate present in receptor is due to phosphorylation by other protein kinases. The kinetics of the phosphorylation of the L-tyrosine- containing peptide were examined by using prephosphorylated insulin receptor/kinase. Prephosphorylation of the receptor was necessary to maximally activate the kinase and to linearize the initial velocity of the peptide phosphorylation reaction. The data obtained rule out a ping-pong mechanism and are consistent with a random-order rapid-equilibrium mechanism for the phosphorylation of this peptide substrate. Additional experiments demonstrated that the autophosphorylated insulin receptor was not able to transfer the preincorporated phosphate to the synthetic peptide substrate. Thus, the insulin receptor/kinase catalyzes the reaction via a mechanism that does not involve transfer of phosphate from a phosphotyrosine-containing enzyme intermediate. Insulin has been shown to elicit its numerous biological effects by interacting with a specific cell-surface receptor [for a review, see Goldfine (1981)l. Work from a number of laboratories has demonstrated that the insulin receptor is a tetramer composed of two CY subunits with a molecular weight of about 135 000 and two zyxwvutsrqp /3 subunits with a molecular weight of about 95 000. The tetrameric structure is held together by disulfide bonds (Jacobs et al., 1979; Massagug et al., 1980; Pilch & Czech, 1980). Kasuga et al. (1982a) first demonstrated that the insulin receptor was associated with a tyrosine protein kinase activity. Since that time, a number of approaches have been utilized to demonstrate that the insulin receptor is itself the tyrosine protein kinase. We (Pike et al., 1986) and others (Petruzelli et al., 1984) have shown that insulin-stimulated kinase activity and insulin binding activity copurify following chromatography on wheat germ lectin-agarose and insulin-agarose columns. In addition, the kinase can be precipitated with antibodies directed against the insulin receptor (Kasuga et al., 1983; Zick et al., 1983). Furthermore, the 95-kilodalton (kDa)' subunit of the insulin receptor can be affinity labeled with azido-ATP (Roth & Cassel, 1983; Shia & Pilch, 1983) or oxidized ATP (Van Obberghen et al., 1983), indicating that the receptor possesses a binding site for this nucleotide as would be expected if it were a protein kinase. Finally, a cDNA for the insulin receptor has been cloned, and the translated amino acid se- quence indicates that the insulin receptor is closely related to the EGF receptor and pp6OSrc, known protein tyrosine kinases, and is also homologous with protein serine kinases, including the CAMP-dependent protein kinase (Ullrich et al., 1985). * Correspondence should be addressed to this author. *Present address: Division of Endocrinology, Stanford University School of Medicine, Stanford, CA 94305. 0006-2960/87/0426-1428$01 .SO10 Until recently, most studies of the insulin receptor have focused on its capacity to recognize and bind insulin and have not examined the enzymatic properties of the kinase activity in detail. For example, the kinase is known to phosphorylate tyrosine residues which are preceded by acidic amino acids (Stadtmauer & Rosen, 1983, 1986), but the specificity of the enzyme for the hydroxyamino acid acceptor has not been established. Furthermore, it has been shown that the insulin receptor/kinase is autophosphorylated and that this auto- phosphorylation stimulates the tyrosine kinase activity of the receptor (Rosen et al., 1983). However, no kinetic analyses have been performed to determine whether auto- phosphorylation produces a required intermediate for the phosphorylation of exogenous substrates. Because the tyrosine kinase activity of this receptor probably mediates many of the intracellular effects of insulin, a clarification of the properties of this enzyme is necessary to the understanding of the process of signal transduction. In this report, we use synthetic peptides to demonstrate that affinity-purified insulin receptor/kinase shows an essentially absolute specificity for tyrosine as the phosphate acceptor. We also present data suggesting that the phosphorylation of peptide substrates occurs via a random-order rapid-equilibrium mechanism and rule out the possibility that an auto- phosphorylated tyrosine serves as a phosphate donor in the phosphorylation of exogenous substrates. EXPERIMENTAL PROCEDURES Materials. Affinity-purified insulin receptors were prepared as described by Pike et al. (1986). Porcine insulin was pur- ' Abbreviations: App(NH)p, 5'-adenylyl imidodiphosphate; EGF, epidermal growth factor; kDa, kilodalton(s); DTT, dithiothreitol. zyxwvutsrqpo 0 1987 American Chemical Society