The Signaling Adapter FRS-2 Competes with Shc for Binding to the Nerve Growth Factor Receptor TrkA

Abstract

We have isolated a human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target and shown that it is tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, we demonstrate that the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclindependent kinase substrate p13 suc1 , and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation.

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

“…While not all of these intracellular targets directly interact with TrkA, several studies indicate that Tyr 499 is essential to the phosphorylation and receptor binding of the PTB domains of Shc (Obermeier et al, 1993;Stephens et al, 1994) and FRS-2 (Meakin et al, 1999). As discussed above, Tyr 794 is essential for the phosphorylation and binding of PLCg-1 and the soluble tyrosine kinase, CHK (Yamashita et al, 1999).…”

“…NGF stimulation of PC12 cells induces the tyrosine phosphorylation of SNT/FRS-2 (80 ± 90 kDa), a di erentiation-speci®c target of NGF and FGF receptors (Rabin et al, 1993;Kouhara et al, 1997;Meakin et al, 1999). We generated a polyclonal antibody to FRS-2 and, as shown in Figure 10a, demonstrate that it immunoprecipitates tyrosine phosphorylated FRS-2 from NGF-stimulated B5 cells (Meakin et al, 1997).…”

“…Upon stimulation, TrkA activates numerous intracellular signaling molecules including the Shc adapter proteins, Grb-2/Sos, Ras, Map kinase (MAPK), phosphatidylinositol-3 kinase, src, phospholipase Cg-1, tyrosine phosphatases such as SH-PTP-1, SH-PTP-2 (Kaplan and Miller, 1997), the Csk homologous tyrosine kinase, CHK (Yamashita et al, 1999), and the more recently described signaling adapters, rAPS, SHB (Qian et al, 1998) and FRS-2 (Meakin et al, 1999). As stated above, TrkA can stimulate a number of di erential responses in cells including mitogenesis in non-neuronal cells, di erentiation and survival in neurons as well as di erentiation and apoptosis in neuronal tumors such as neuroblastomas and medulloblastomas respectively.…”

“…Consistent with the model of RTK activation, NGF binding to TrkA stimulates receptor dimerization and trans-phosphorylation of the activation loop tyrosines which, in turn, induce allosteric changes to stabilize the kinase (Heldin, 1995). TrkA then activates a variety of intracellular signaling molecules such as the Shc adapter proteins, Grb-2, Sos, phospholipase Cg-1 (PLCg-1), phosphatidylinositol 3-kinase (PI-3 kinase), Akt, Ras, mitogen-activated protein (MAP) kinases [or extracellular signal-regulated kinases (Erks)], c-yes, protein tyrosine phosphatase-1 C, the SH2-domain containing tyrosine phosphatase SH-PTP-2 ( Kaplan and Miller, 1997), the CSK homologous tyrosine kinase, CHK (Yamashita et al, 1999), and the adapter proteins FRS-2 (Kouhara et al, 1997;Meakin et al, 1999), SH2-B and rAPS (Qian et al, 1998).…”

“…The catalytic core tyrosines (Tyr 679 , Tyr 683 and Tyr 684 ) are essential to receptor phosphorylation and activation while Tyr 499 and Tyr 794 are required for the tyrosine phosphorylation of Shc/ FRS-2 and PLCg-1/CHK, respectively, and serve as the docking sites for their high-a nity binding to TrkA (Obermeier et al, 1994;Stephens et al, 1994;Meakin et al, 1999;Yamashita et al, 1999).…”