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 extra cellular domain of the beta-PDGF receptor and the intracellular and tr ansmembrane 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 i nositol phospholipid 3-kinase and phospholipase C-gamma 1, respectivel y. The strong and sustained activation of p42 and p44 mitogen-activate d-protein kinases induced by PDGF-B/B in PC12/PT-R cells was unaffecte d by mutation of Tyr785 or Tyr751 to phenylalanine, but was smaller an d transient after mutation of Tyr490, and almost abolished by the doub le mutation of Tyr490 and Tyr785. Mutation of Tyr490 reduced by 70% th e PDGF-induced increase in inositol phospholipid 3-kinase activity imm unoprecipitated from cell extracts with antiphosphotyrosine monoclonal antibodies and greatly suppressed the PDGF-induced increase in the in tracellular products of inositol phospholipid 3-kinase, while mutation of Tyr751 or Tyr785 had no effect. Mutation of Tyr785 (but not mutati on of Tyr490 or Tyr751) abolished PDGF-stimulated hydrolysis of phosph atidylinositol 4,5-bisphosphate. Mutation of Tyr490, alone or in combi nation with mutation of Tyr751 and Tyr785, had no effect on the PDGF-i nduced 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 phosphorylat ion of Tyr490 plays a major role in the activation of inositol phospho lipid 3-kinase and formation of 3-phosphorylated inositol lipids and c onfirm that the phosphorylation of Tyr 785 triggers the activation of phospholipase C-gamma 1 in vivo. (2) Tyr490 phosphorylation (but not i nositol phospholipid 3-kinase activation) is also required for strong and sustained activation of mitogen-activated-protein kinase and neuro nal differentiation, while the smaller and more transient activation o f mitogen-activated-protein kinase, produced by the activation of phos pholipase C-gamma 1 is insufficient to trigger the neuronal differenti ation of PT-R cells. (3) Inositol phospholipid 3-kinase is required fo r the activation of p70(S6K), but only a small increase in inositol ph ospholipid 3-kinase activity and the level of 3-phosphorylated inosito l lipids is required for maximal p70(S6K) activation.