Distinct Tyrosine Autophosphorylation Sites Negatively and Positively Modulate Neu-Mediated Transformation

Abstract

A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Rasdependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.The neu/c-ErbB-2 gene encodes a 185-kDa transmembrane receptor tyrosine kinase that is a member of the epidermal growth factor receptor (EGFR) family (4,15,76). Elevated expression of the EGFR family members has been implicated in the etiology of human ovarian and breast cancers (reviewed in references 31 and 41). In particular, amplification and consequent overexpression of the neu receptor tyrosine kinase have been observed in a large proportion of primary human breast cancers (58,64,65). Consistent with these observations, expression of either wild-type neu or a constitutively active mutant (V664E, neuNT [3,5]) in the mammary epithelia of transgenic mice results in the induction of metastatic mammary tumors (11,27,47). The induction of mammary tumors in the wild-type neu transgenic mice correlates with elevated

Copine-III interacts with ErbB2 and promotes tumor cell migration

“…Copine-III does not possess an SH2-or a phosphotyrosine-pTyr binding domain. Shc has both domains and has also been found associated with pTyr1248 (Dankort et al, 1997;Schulze et al, 2005) (Figure 1). Shc was detected in Copine-III IPs from lysates of T47D cells, and Shc levels increased in response to HRG (Figure 5d).…”

“…Tyr1028 couples to a pathway implicated in negative regulation of NeuT transforming potential, whereas the four other sites have a potentiating effect. Tyr1144, Tyr1201 and Tyr1227 signal through the Ras/Erk pathway by binding Grb2, Crk and Shc (Akiyama et al, 1991;Dankort et al, 1997Dankort et al, , 2001, which also binds Tyr1253 (Schulze et al, 2005). The role of pTyr residues has also been examined in migration assays.…”

Signal transduction in mammary tumorigenesis: a transgenic perspective

“…Shc proteins are thought to promote Ras activation by indirectly recruiting Grb2 Rozakis-Adcock et al, 1992) through tyrosine phosphorylation of sites conforming to consensus Grb2 SH2 binding sites (Gotoh et al, 1997;Salcini et al, 1994;Songyang et al, 1994). Ras activity is required for e cient ErbB-2 (Ben- Levy et al, 1994;Dankort et al, 1997) and PyV-MT-(Jelinek and Hassell, 1992) mediated signaling.…”

“…While none of the ®ve known ErbB-2 tyrosine phosphorylation sites are required for transformation (Segatto et al, 1990;Mikami et al, 1992;Ben-Levy et al, 1994;Dankort et al, 1997), four sites (Y1144, Y1201, Y1226/7, Y1253) can independently mediate a transforming signal and require Ras activity to induce DNA synthesis (Dankort et al, 1997). Of these Y1144 and Y1226/7 respectively bind Grb2 and Shc and interaction directly correlates with transformation potential, thus providing a molecular mechanism for Ras activation (Dankort and Muller, unpublished observations).…”

Signal transduction pathways activated and required for mammary carcinogenesis in response to specific oncogenes

“…A role for the mitogen activated protein kinases (MAPKs or Erks) in transformation by the ErbB2 receptor has been established by showing that overexpression of ErbB2 is associated with a considerably increased Erk/ MAP kinase activity in breast cancer cell lines (Janes et al, 1994). Furthermore, the autophosphorylation sites in the ErbB2/Neu receptor that allow coupling to Ras and the Erk/MAP kinase pathway appear to be very important for cell transformation (Ben-Levy et al, 1994;Dankort et al, 1997). The role of signal transduction pathways in transformation mediated by ligands of the EGF family is not well understood at present, but studies have shown that exogenously added EGF and heregulin/NDF can induce activation of the Erk/MAP kinases, Jnk/SAP kinases (stress activated protein kinases) and phosphatidyl inositol 3-kinase in mammary epithelial cell lines.…”