A Peptide-based Protein-tyrosine Phosphatase Inhibitor Specifically Enhances Insulin Receptor Function in Intact Cells*

3S-peptide-I is a synthetic tris-sulfotyrosyl dodecapeptide corresponding to the major site of insulin receptor autophosphorylation that potently inhibits dephosphorylation of the insulin receptor in a cell-free system and in digitonin-permeabilized Chinese hamster ovary (CHO) cells overexpressing the human insulin receptors (CHO/HIRc cells) (Liotta, A. S., Kole, H. K., Fales, H. M., Roth, J., and Bernier, M. (1994) J. Biol. Chem. 269, 22996-23001). In the present study, we found that 3S-peptide-I was not capable of inhibiting dephosphorylation of the epidermal growth factor (EGF) receptors in digitonin-permeabilized CHO cells that overexpress human EGF receptors (CHO/EGF-R cells). Moreover, the addition of a N-stearyl derivative of 3S-peptide-I to intact CHO/HIRc cells caused a concentration-dependent increase in insulin-stimulated phosphorylation of the insulin receptor, with a maximum effect (∼2.7-fold) at 50 μM. In contrast, ligand-stimulated EGF receptor phosphorylation in CHO/EGF-R cells was not affected by the presence of stearyl 3S-peptide-I. Furthermore, treatment of CHO/HIRc cells with this N-stearyl peptide led to a significant enhancement of the insulin-induced association of phosphatidylinositol (PI) 3-kinase activity with insulin receptor substrate 1 and the activation of mitogen-activated protein kinase. However, stearyl 3S-peptide-I had no effect on the EGF-stimulated activation of PI-3-kinase and mitogen-activated protein kinase in CHO/EGF-R cells. These data indicate that this tris-sulfotyrosyl dodecapeptide selectively enhances insulin signal transduction by specifically inhibiting dephosphorylation of the insulin receptor in intact cells.

[1]  J. Meyerovitch,et al.  Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. Characterization and mode of action. , 1987, The Journal of biological chemistry.

[2]  P. Roller,et al.  Potent inhibition of insulin receptor dephosphorylation by a hexamer peptide containing the phosphotyrosyl mimetic F2Pmp. , 1994, Biochemical and biophysical research communications.

[3]  P. H. Seeburg,et al.  Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes , 1985, Nature.

[4]  D. Brautigan,et al.  Specificity of protein phosphotyrosine phosphatases. Comparison with mammalian alkaline phosphatase using polypeptide substrates. , 1985, The Journal of biological chemistry.

[5]  R. Henry,et al.  Skeletal muscle protein tyrosine phosphatase activity and tyrosine phosphatase 1B protein content are associated with insulin action and resistance. , 1994, The Journal of clinical investigation.

[6]  R. Bradshaw,et al.  Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[7]  E. Krebs,et al.  Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase. , 1991, The Journal of biological chemistry.

[8]  C. Kahn,et al.  Insulin action and the insulin signaling network. , 1995, Endocrine reviews.

[9]  L. Cantley,et al.  Activation of phosphatidylinositol 3-kinase by insulin. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Dixon,et al.  Protein tyrosine phosphatase substrate specificity: size and phosphotyrosine positioning requirements in peptide substrates. , 1994, Biochemistry.

[11]  R. Epand,et al.  Insulin receptor autophosphorylation and signaling is altered by modulation of membrane physical properties. , 1995, Biochemistry.

[12]  A. Weiss,et al.  Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Ing Ts.  Phosphotyrosyl-protein phosphatases. II. Identification and characterization of two heat-stable protein inhibitors. , 1989 .

[14]  F. Casanueva,et al.  Regulation of epidermal-growth-factor-receptor signal transduction by cis-unsaturated fatty acids. Evidence for a protein kinase C-independent mechanism. , 1991, The Biochemical journal.

[15]  A. Shisheva,et al.  Role of cytosolic tyrosine kinase in mediating insulin-like actions of vanadate in rat adipocytes. , 1993, The Journal of biological chemistry.

[16]  G. J. Sale,et al.  Site-specific dephosphorylation and deactivation of the human insulin receptor tyrosine kinase by particulate and soluble phosphotyrosyl protein phosphatases. , 1991, The Biochemical journal.

[17]  C. Kahn,et al.  Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. , 1982, Science.

[18]  W. Merlevede,et al.  Isolation and characterization of a tyrosyl phosphatase activator from rabbit skeletal muscle and Xenopus laevis oocytes. , 1990, Biochemistry.

[19]  B. Franza,et al.  Multi‐site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation. , 1993, The EMBO journal.

[20]  P. Seeburg,et al.  Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells , 1984, Nature.

[21]  T. Sturgill,et al.  Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[22]  A. Saltiel,et al.  Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein kinase by insulin. , 1994, The Journal of biological chemistry.

[23]  B. Neel,et al.  The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence , 1992, Cell.

[24]  N. Perrimon,et al.  corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso , 1992, Cell.

[25]  P. Vicario,et al.  The insulin-mimetic effect of vanadate is not correlated with insulin receptor tyrosine kinase activity nor phosphorylation in mouse diaphragm in vivo. , 1989, Endocrinology.

[26]  C. Kahn,et al.  The insulin signaling system. , 1994, The Journal of biological chemistry.

[27]  M. White,et al.  Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1 , 1993, Molecular and cellular biology.

[28]  D. D. Shock,et al.  Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells. , 1994, Biochemistry.

[29]  M. Czech,et al.  Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulin-treated adipocytes. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Casnellie,et al.  The insulin-like effect of vanadate on lipolysis in rat adipocytes is not accompanied by an insulin-like effect on tyrosine phosphorylation. , 1989, Endocrinology.

[31]  S. Sabatini,et al.  Biochemistry and pathophysiology of vanadium. , 1994, Nephron.

[32]  P. Lipsky,et al.  Promotion of human T lymphocyte activation and proliferation by fatty acids in low density and high density lipoproteins. , 1986, The Journal of biological chemistry.

[33]  G. J. Sale,et al.  Dephosphorylation of insulin-receptor autophosphorylation sites by particulate and soluble phosphotyrosyl-protein phosphatases. , 1990, The Biochemical journal.

[34]  B. Geiger,et al.  Hepatic tyrosine-phosphorylated proteins identified and localized following in vivo inhibition of protein tyrosine phosphatases: effects of H2O2 and vanadate administration into rat livers , 1993, Molecular and Cellular Endocrinology.

[35]  J. Dixon,et al.  Substrate specificity of the protein tyrosine phosphatases. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  C. Walsh,et al.  Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTPβ, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors , 1993, Protein science : a publication of the Protein Society.

[37]  R. Klausner,et al.  Lipid domains in membranes. Evidence derived from structural perturbations induced by free fatty acids and lifetime heterogeneity analysis. , 1980, The Journal of biological chemistry.

[38]  B. Goldstein,et al.  Insulin receptor protein-tyrosine phosphatases. Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain. , 1992, The Journal of biological chemistry.

[39]  C. Walsh,et al.  Purification and characterization of a soluble catalytic fragment of the human transmembrane leukocyte antigen related (LAR) protein tyrosine phosphatase from an Escherichia coli expression system. , 1991, Biochemistry.

[40]  A. Ullrich,et al.  Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. , 1993, Science.

[41]  D. Banville,et al.  Inhibition of the activity of protein tyrosine phosphate 1C by its SH2 domains. , 1993, Biochemistry.

[42]  C. Kahn,et al.  Hepatic phosphotyrosine phosphatase activity and its alterations in diabetic rats. , 1989, The Journal of clinical investigation.

[43]  C. Bogardus,et al.  Abnormal Regulation of Protein Tyrosine Phosphatase Activities in Skeletal Muscle of Insulin-Resistant Humans , 1991, Diabetes.

[44]  A. Saltiel,et al.  Protein-tyrosine-phosphatase SHPTP2 is a required positive effector for insulin downstream signaling. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. Bernier,et al.  A synthetic tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain inhibits tyrosine dephosphorylation of the insulin receptor in situ. , 1994, The Journal of biological chemistry.

[46]  T. Burke,et al.  Phosphonate inhibitors of protein-tyrosine and serine/threonine phosphatases. , 1995, The Biochemical journal.

[47]  G. J. Sale,et al.  Regulation of an hepatic low-M(r) membrane-associated protein-tyrosine phosphatase. , 1993, The Biochemical journal.

[48]  D. Brautigan,et al.  Activation of membrane protein-tyrosine phosphatase involving cAMP- and Ca2+/phospholipid-dependent protein kinases. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[49]  S. Shoelson,et al.  Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction. , 1994, The Journal of biological chemistry.

[50]  R. Dixon,et al.  The phosphotyrosine phosphatase inhibitor vanadyl hydroperoxide induces morphological alterations, cytoskeletal rearrangements and increased adhesiveness in rat neutrophil leucocytes. , 1993, Journal of cell science.

[51]  R. Aebersold,et al.  Sequential dephosphorylation of a multiply phosphorylated insulin receptor peptide by protein tyrosine phosphatases. , 1992, Biochemistry.

[52]  G. Swarup,et al.  Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. , 1982, Biochemical and biophysical research communications.

[53]  William Arbuthnot Sir Lane,et al.  Role of IRS-2 in insulin and cytokine signalling , 1995, Nature.

[54]  D. Barford,et al.  Protein-Tyrosine Phosphatase Inhibition by a Peptide Containing the Phosphotyrosyl Mimetic, L-O-Malonyltyrosine (L-OMT) , 1995 .

[55]  B. Goldstein,et al.  Increased abundance of the receptor-type protein-tyrosine phosphatase LAR accounts for the elevated insulin receptor dephosphorylating activity in adipose tissue of obese human subjects. , 1995, The Journal of clinical investigation.

[56]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[57]  C. Walsh,et al.  NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase , 1992, Protein science : a publication of the Protein Society.

[58]  B. Goldstein,et al.  Insulin Receptor Signaling Is Augmented by Antisense Inhibition of the Protein Tyrosine Phosphatase LAR (*) , 1995, The Journal of Biological Chemistry.