Autophosphorylation of Activation Loop Tyrosines Regulates Signaling by the TRK Nerve Growth Factor Receptor

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 C␥-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 C␥-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.For many growth factors, ligand-mediated activation of receptor tyrosine kinases (RTKs) 1 is the first essential step leading to biological responses (1, 2). Nerve growth factor (NGF) and other neurotrophins bind to and activate particular members of the Trk receptor tyrosine kinase family (3, 4); NGF binds to and activates TRKA (5, 6). The pheochromocytomaderived PC12 cell line (7) has been highly useful for studying NGF signal transduction and its molecular mechanisms of action. PC12 cells respond to NGF by undergoing neuronal differentiation, survival in serum-free medium, cell hypertrophy, and regulation of numerous biochemical markers (8). 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). Activation of these signal transduction molecules culminates in downstream signaling events, including the induction of immediate early and late genes (10, 11).As with other ligands and their cognate RTKs (1, 2), NGF appears to initiate signaling through TrkA by the sequential steps of dimerization, trans-autophosphorylation on tyrosine residues and kinase activation, followed by binding and tyrosine phosphorylation of substrates (9,12,13). In vitro studies indicate that TRKA autophosphorylates at tyrosines 490, 670, 674, 675, and 785 (14, 15). To analyze the significance of thes...

Acidic substitution of the activation loop tyrosines in TrkA supports nerve growth factor-independent cell survival and neuronal differentiation

“…Moreover, since hydrophilic homologues such as AsnGln or GlnAsn do not induce NGF-independent kinase activation or neurite outgrowth (Figures 2 and 5), it is clear that it is the negative charge of residues 683 and 684, and not their hydrophilic nature, that is required for the release of auto-inhibition and subsequent activation. This may re¯ect a requirement for interaction between negatively charged residues in the activation loop with the positively charged COOHterminal tail in order to stabilize the kinase consistent with a model previously suggested by Cunningham et al (1997).…”

“…This discrepancy is surprising and not well understood but presumably re¯ects either absolute di erences in the activation of intracellular substrates, changes in the kinetics of substrate activation and/or changes in the stoichiometric relationships of intracellular targets. Non-linearity between the level of TrkA phosphorylation and neuritogenesis has previously been observed (Cunningham et al, 1997).…”

“…Tyr 684 in TrkA, the positional equivalent of Tyr 1163 in the IRK, is thought to stabilize an active conformation. Consistent with this model, phenylalanine mutagenesis of the catalytic core tyrosines in TrkA, TrkB, the IRK, the IGF-1 receptor, the FGF receptor and Met e ectively inactivates kinase activity (Ellis et al, 1986;Wilden et al, 1992;Li et al, 1994;Longati et al, 1994;Hernandez-Sanchez et al, 1995;Stannard et al, 1995;Cunningham et al, 1997;McCarty and Feinstein, 1998).…”

“…For comparison, mutants containing PhePhe, AsnGln or GlnAsn were also assayed ( Figure 2d) and found to be only 5 ± 10% as active as wild type TrkA. The PhePhe substitution has previously been shown to functionally inactivate TrkA (Cunningham et al, 1997).…”

Specific TrkA survival signals interfere with different apoptotic pathways

“…Tyrosine residues Y670, Y674 and Y675 residing within the receptor activation loop' are not only important for the intrinsic activity of the receptor but regulate the phosphorylation of the two tyrosine residues outside the catalytic domain, Y490 and Y785, therefore controlling speci®c signalling pathways (Mitra, 1991;Cunningham et al, 1997). Although the autophosphorylation of these`activation loop' residues should play a role in TrkA survival signalling it is not su cient to elicit an anti-apoptotic e ect because TrkA receptors mutated only at the two tyrosines outside the kinase domain (TrkA YY490/785FF) did not show any survival response.…”