Abstract
Many receptor tyrosine kinases possess an "activation loop" containing three similarly placed tyrosine autophosphorylation sites. To examine their roles in the TRK NGF receptor, these residues (Tyr-670, Tyr-674, and Tyr-675) were mutated singly and in all combinations to phenylalanine and stably expressed in Trk-deficient PC12nnr5 cells. All mutant receptors showed significantly diminished nerve growth factor (NGF)-stimulated autophosphorylation, indicating impaired catalytic activity. NGF-induced neurite outgrowth exhibited dose-responsive behavior when transfectants were compared by relative receptor expression and exhibited a functional hierarchy: wild type > Y670F >/= Y674F >> Y675F >/= YY670/674FF = YY670/675FF >> YY674/675FF > YYY670/674/675FFF. NGF-induced tyrosine phosphorylation of Shc, ERKs, and SNT and immediate early gene inductions generally paralleled neurogenic potential. However, activation of phosphatidylinositol 3'-kinase and tyrosine phosphorylation of phospholipase Cgamma-1 was essentially abolished. The latter effect appears due to selective inability of the mutated TRKs to autophosphorylate the tyrosine residue (Tyr-785) required for binding phospholipase Cgamma-1 and indicates that the "activation loop" tyrosines participate in NGF-dependent changes in receptor conformation. Our findings stress the importance that expression levels play in assessing the consequences of receptor mutations and that all three activation loop tyrosines have roles regulating both overall and specific NGF-mediated signaling through TRK.
Highlights
Ligand-mediated activation of receptor tyrosine kinases (RTKs)1 is the first essential step leading to biological responses [1, 2]
These and wild type (WT) TRK were cloned into the pLNCX retrovirus vector [54], and the resultant recombinant retroviruses were used to infect the Trk-deficient PC12-derived cell line, PC12nnr5 [16, 52]
This was achieved by mutating these tyrosines singly, and in all combinations, and examining the signaling properties of each mutant receptor when stably expressed at various levels in PC12nnr5 cells that lack endogenous Trk
Summary
Ligand-mediated activation of receptor tyrosine kinases (RTKs) is the first essential step leading to biological responses [1, 2]. NGF induces rapid tyrosine phosphorylation of TrkA and consequent phosphorylation and activation of signal transduction components including Shc, extracellular signal-regulated kinases (ERKs), phosphatidylinositol 3Ј-kinase (PI3ЈK), phospholipase C ␥-1 (PLC␥-1), and SNT [9]. To analyze the significance of these phosphorylation events in NGF signaling, we have introduced TRKA receptors modified at these residues into the mutant PC12 cell line, PC12nnr, that expresses no detectable endogenous TrkA [16, 17] This analysis reveals that phosphotyrosines 490 and 785 are required for binding and activation of Shc and PLC ␥-1, respectively [14, 15, 18]. Activation Loop Phosphorylation and TRK Signaling was suggested that ligand-promoted phosphorylation of activation loop tyrosines induces movement of the loop away from the catalytic core to permit efficient kinase activation
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