Phosphotyrosine Residues in the Nerve‐Growth‐Factor Receptor (Trk‐A)

Abstract

-EJB 95 1044/1 PC12 cells, which lack platelet derived-growth-factor (PDGF) receptors, have been stably transfected with a chimaera consisting of the extracellular domain of the p-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-yl , respectively. The strong and sustained activation of p42 and p44 mitogenactivated-protein kinases induced by PDGF-B/B in PC12/PT-R cells was unaffected by mutation of Tyr78.5 or Tyr751 to phenylalanine, but was smaller and transient after mutation of Tyr490, and almost abolished by the double mutation of Tyr490 and Tyr78.5. Mutation of Tyr490 reduced by 70% the PDGFinduced 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 Tyr7.51) 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 (~7 0 "~) .However, the activation of p70ShK by PDGF (or nerve growth factor), but not the activation of mitogen-activatedprotein 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-yl 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-yl is insufficient to trigger the neuronal differentiation of PT-R cells. (3) Inositol phospholipid 3-kinase is required for the activation of p70SfiK, but only a small increase in inositol phospholipid 3-kinase activity and the level of 3-phosphorylated inositol lipids is required for maximal p70SbK activation.Keywords: PC12 cells ; nerve growth factor; platelet-derived growth factor; inositol phospholipid 3-kinase ; mitogen-activated-protein kinase.Nerve growth factor (NGF), a neurotrophic factor essential for the survival and development of sympathetic neurones, causes PC12 cells to differentiate from immature chromaffin cells to a sympathetic neuron-like phenoty...

Specific TrkA survival signals interfere with different apoptotic pathways

“…This result was con®rmed using two independent stable cell lines, one of which (C7) expresses equal and the other (C11) expressing higher levels of TrkA Y490F protein compared to the wt-TrkA clone (G3) (Figure 3b and c). In contrast to TrkA Y490F, TrkA Y785F e ectively activates Akt/PKB, con®rming earlier reports, indicating that residue Y490 drives PI3-kinase activation by NGF-stimulated TrkA (Baxter et al, 1995); Figure 3c The phosphorylation and expression levels of the TrkA mutant clones C7, C11 (both Y490F) and of clone B30 (Y785F) were monitored as described in Materials and methods. Clone C7 expresses levels of TrkA Y490F comparable to those of the wt-clone G3, whereas clones C11 and B30 express about four times higher receptor levels than clone C7.…”

“…Earlier in vitro studies suggested that the p85 subunit of PI3-kinase associates with NGF-stimulated TrkA through tyrosine residue Y751 (Obermeier et al, 1993b). However, recent studies using mutant TrkA receptors indicate that the activation of PI3-kinase depends in the main on phosphorylation of tyrosine Y490, the SHC binding site in human TrkA (Baxter et al, 1995). The PI3-kinase targets S6-kinase (Weng et al, 1995) and Akt/PKB (Burgering and Co er, 1995;Franke et al, 1995) Figure 2b, respectively).…”

Nerve growth factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoinositide 3-OH kinase

“…The e ect of mutating Y490 and Y785 on the induction of PI 3-kinase activity by Trk in intact cells has been previously reported (Baxter et al, 1995). Using chimeric PDGF receptor/Trk molecules expressed in PC12 cells, it was shown that Y490 was absolutely required for the elevation of PI(3,4)P 2 by PDGF.…”

“…In normal PC12 cells treated with NGF, PI 3-kinase activity cannot be found in Trk immunoprecipitates (Carter and Downes, 1992;Solto et al, 1992). In vitro, a phosphorylated peptide corresponding to the site around amino acid Y751 of Trk interacts with the p85 subunit of the PI 3-kinase, although it appears that this site is not actually phosphorylated in vivo under physiological circumstances (Baxter et al, 1995;Obermeier et al, 1993b). However, it is known that NGF is able to stimulate both the formation of Ras.GTP (Nakafuku et al, 1992;Qiu and Green, 1991) and 3' phosphorylated phosphoinositides (Baxter et al, 1995;Carter and Downes, 1992) in intact PC12 cells.…”

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

“…In this respect, PI-3 kinase appears to be functionally downstream of Ras (Rodriguez-Viciana et al, 1994;Hallberg et al, 1998). In fact, a Shc-minus human TrkA receptor exhibits a signi®cant loss of NGF-activated PI-3 kinase activity in vitro (Baxter et al, 1995;Hallberg et al, 1998). However, substitution of the ®rst catalytic tyrosine, Tyr 670 (human TrkA), also signi®cantly decreases PI-3 kinase activity, with minimal e ects on Shc-dependent pathways (Cunningham et al, 1997), suggesting that there is more than one route to PI-3 kinase activation.…”