PHOSPHOTYROSINE RESIDUES IN THE NERVE-GROWTH-FACTOR RECEPTOR (TRK-A) - THEIR ROLE IN THE ACTIVATION OF INOSITOLPHOSPHOLIPID METABOLISM AND PROTEIN-KINASE CASCADES IN PHEOCHROMOCYTOMA (PC12) CELLS

PC12 cells, which lack platelet derived-growth-factor (PDGF) receptors, have been stably transfected with a chimaera consisting of the extracellular domain of the beta-PDGF receptor and the intracellular and transmembrane domains of the nerve-growth-factor receptor Trk-A (termed PT-R). Mutation of the Trk-A residue Tyr490 to phenylalanine prevents the association with Shc, while similar mutations at Tyr751 or Tyr785 are reported to prevent interaction of Trk-A with the p85 subunit of inositol phospholipid 3-kinase and phospholipase C-gamma 1, respectively. The strong and sustained activation of p42 and p44 mitogen-activated-protein kinases induced by PDGF-B/B in PC12/PT-R cells was unaffected by mutation of Tyr785 or Tyr751 to phenylalanine, but was smaller and transient after mutation of Tyr490, and almost abolished by the double mutation of Tyr490 and Tyr785. Mutation of Tyr490 reduced by 70% the PDGF-induced increase in inositol phospholipid 3-kinase activity immunoprecipitated from cell extracts with antiphosphotyrosine monoclonal antibodies and greatly suppressed the PDGF-induced increase in the intracellular products of inositol phospholipid 3-kinase, while mutation of Tyr751 or Tyr785 had no effect. Mutation of Tyr785 (but not mutation of Tyr490 or Tyr751) abolished PDGF-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. Mutation of Tyr490, alone or in combination with mutation of Tyr751 and Tyr785, had no effect on the PDGF-induced activation of p70 S6 kinase (p70(S6K)). However, the activation of p70(S6K) by PDGF (or nerve growth factor), but not the activation of mitogen-activated-protein kinase, was prevented by two structurally unrelated inhibitors of inositol phospholipid 3-kinase, wortmannin or LY294002. Our results demonstrate the following: (1) the phosphorylation of Tyr490 plays a major role in the activation of inositol phospholipid 3-kinase and formation of 3-phosphorylated inositol lipids and confirm that the phosphorylation of Tyr 785 triggers the activation of phospholipase C-gamma 1 in vivo. (2) Tyr490 phosphorylation (but not inositol phospholipid 3-kinase activation) is also required for strong and sustained activation of mitogen-activated-protein kinase and neuronal differentiation, while the smaller and more transient activation of mitogen-activated-protein kinase, produced by the activation of phospholipase C-gamma 1 is insufficient to trigger the neuronal differentiation of PT-R cells. (3) Inositol phospholipid 3-kinase is required for the activation of p70(S6K), but only a small increase in inositol phospholipid 3-kinase activity and the level of 3-phosphorylated inositol lipids is required for maximal p70(S6K) activation.