Differential Utilization of ShcA Tyrosine Residues and Functional Domains in the Transduction of Epidermal Growth Factor-induced Mitogen-activated Protein Kinase Activation in 293T Cells and Nerve Growth Factor-induced Neurite Outgrowth in PC12 Cells

Abstract

By transient expression of both truncated forms of p52 SHCA and those with point mutations in 293T cells, it has been shown that, in addition to Tyr-317, Tyr-239/240 is a major site of phosphorylation that serves as a docking site for Grb2⅐Sos1 complexes. In addition, analysis of epidermal growth factor (EGF)-induced activation of mitogen-activated protein kinase in 293T cells showed that the overexpression Shc SH2 or phosphotyrosine binding (PTB) domains of ShcA alone has a more potent negative effect than the overexpression of the forms of ShcA lacking Tyr-317 or Tyr 239/240 or both. In transiently transfected PC12 cells, the ShcA PTB domain and tyrosine phosphorylation in the CH1 domain, especially on Tyr-239/240, are crucial for mediating nerve growth factor (NGF)-induced neurite outgrowth. These findings suggest that the EGF and NGF (TrkA) receptor can utilize Shc in different ways to promote their activity. For EGF-induced mitogen-activated protein kinase activation in 293T cells, both Shc PTB and SH2 domains are essential for optimal activation, indicating that a mechanism independent of Grb2 engagement with Shc may exist. For NGF-induced neurite outgrowth in PC12 cells, Shc PTB plays an essential role, and phosphorylation on Tyr-239/240, but not on Tyr-317, is required.Activation of the Ras/MAP 1 kinase pathway, triggering proliferation and differentiation, is one of the major early events induced by receptor tyrosine kinases (reviewed in Ref. 1). Grb2 and Sos1 have been linked to the mediation of this activation (2, 3). Grb2 is a 23-kDa adaptor protein composed of an SH2-type phosphotyrosine recognition domain, flanked by two SH3 domains that recognize proline-rich sequences (reviewed in Ref. 4). Sos1 is a 170-kDa protein with a guanine nucleotide exchanging domain that can catalyze the conversion of inactive Ras, with bound GDP, into its active form, with bound GTP (reviewed in Ref. 5). Beside its catalytic domain, Sos1 contains a proline-rich region enabling it to couple with Grb2 (6). Activation of Ras can be achieved when cytosolic Sos translocates to the plasma membrane, where cellular Ras normally resides (7). This translocation is mediated by the recruitment of Grb2 onto the intracellular domain of an activated receptor tyrosine kinase, such as the epidermal growth factor receptor (EGFR) (8). Grb2 can dock through its SH2 domain with specific phosphotyrosine residues of the activated EGFR which subsequently allows relocalization of Sos1 to the plasma membrane level (reviewed in Ref. 9). Activated Ras allows a cascade of events starting with the activation of the serine kinase Raf that phosphorylates and activates a MAP kinase kinase (also denoted MEK), which in turn activates MAP kinase by dual phosphorylation on tyrosine and threonine residues (reviewed in Ref. 1).Although the EGFR can utilize Grb2 directly to mediate MAP kinase activation, it can also recruit Grb2 and Sos1 indirectly, which involves the Shc family of adaptor proteins (10, 11). The first member identified and the prototype of th...

Association of the Atypical Protein Kinase C-interacting Protein p62/ZIP with Nerve Growth Factor Receptor TrkA Regulates Receptor Trafficking and Erk5 Signaling

“…With respect to TrkA, it has been shown that Shc is required for this pathway (13,34,38). The failure of Shc to compete for with p62 for binding to TrkA reveals that p62 activates the B pathway independently of this site.…”

The NF-κB-inducing Kinase Induces PC12 Cell Differentiation and Prevents Apoptosis

“…Expression Vectors and Cell Culture-The expression vectors encoding IB␣ S32A/S36A (SS/AA) and the dominant-negative Shc (Y239F/ Y240F/Y317F) mutant have been described elsewhere (25,26). Ecdysone-responsive (EcR) PC12 cells stably expressing WT Myc-NIK and kinase-deficient Myc-NIK (K429A/K430A) under the control of the ecdysone-inducible pIND vector (Invitrogen) were selected with hygromycin (Life Technologies, Inc.).…”

Comparison of the intracellular signaling responses by three chimeric fibroblast growth factor receptors in PC12 cells

“…Lysates from PC12 cell lines, untreated or stimulated with PDGF (30 ng͞ml) at 37°C for 5, 30, and 120 min, were incubated with 10 l of agaroseconjugated polyclonal anti-Erk1 (Santa Cruz Biotechnology) for 3 hr at 4°C. After washing the agarose beads once in lysis buffer and once in kinase buffer, kinase reactions were performed in the presence of 2 mg͞ml myelin basic protein (as substrate), 50 M ATP, and 5 Ci (1 Ci ϭ 37 GBq) [␥-32 P]ATP essentially as described (19).…”