Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential

Abstract

Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises one step in the signaling events mediated by erbB receptors such as p185 neu and EGFR. Previous analysis of p185 neu has indicated that there are at least ®ve tyrosine autophosphorylation sites, Y882, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have speci®cally analysed the e ect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine 882 by phenylalanine signi®cantly decreased the transforming potential of activated, oncogenic p185 neu , although no signi®cant di erence in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185 neu and Y882F were fused with the extracellular domain of the EGF receptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185 neu . These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine 882 residue may be important for p185 neu -mediated transformation by a ecting the enzymatic kinase function of the p185 neu receptor.

Loss of Hsp90 Association Up-Regulates Src-Dependent ErbB2 Activity

“…Upon Tyr877 phosphorylation, the phosphoryl group establishes strong salt bridges with two arginine residues, which we suggest induces a conformational change in the A-loop, enabling it to attain the active conformation. Consistent with our observations, which were made with the human ErbB2 protein, Src dependence has also been observed for the constitutively active rat ErbB2/neu protein (39). Importantly, the amino acid sequence of the A-loop of rat ErbB2/neu is identical to that of human ErbB2, suggesting it may also undergo a conformational change when the corresponding Tyr882 is phosphorylated.…”

“…Tyr882 phosphorylation of the A-loop of rat ErbB2/neu is important for its intrinsic kinase activity (39). We determined whether the analogous Tyr877 is phosphorylated to a higher level in ErbB2-5M than in the wild-type kinase, and we found that it was dramatically increased (Fig.…”

“…Phosphorylation, in tumor cells, on the analogous site in ErbB2, Tyr877, was reported recently (12,27), but the functional implication is not clear. In contrast to ErbB1, data reported for the oncogenic rat protein ErbB2/neu, which carries an activating point mutation in the transmembrane domain, suggest that A-loop phosphorylation is important for intrinsic kinase activity, as mutation of the analogous Tyr882 to phenylalanine compromised kinase activity (39).…”

Sensitivity to pertuzumab (2C4) in ovarian cancer models: cross-talk with estrogen receptor signaling

“…Whereas the significance and functionality of HER2 (Tyr 877 ) phosphorylation is poorly defined (22), phosphorylation of the homologous HER1 (Tyr 845 ) site is nevertheless known to be src mediated (23). Because the homologous site to HER2 (Tyr 877 ) in the activated rat p185 neu protein (HER2 Tyr 882 ) is an autophosphorylation site and mutation of this residue reduces the intrinsic kinase activity of the protein and its transforming potential, this suggests that this tyrosine residue has an important functional role (24). Other phosphorylation sites on HER1 and HER2 were less informative, although the increased phosphorylation status of HER1 (Tyr 1045 ) in OVCAR5 and 41M cells (Fig.…”

Trifunctional antibody ertumaxomab: Non-immunological effects on Her2 receptor activity and downstream signaling

“…32 The Y877 residue, for instance, is located in the activation loop of the Her2 kinase domain. Although the role of Y877-Her2-phosphorylation is the subject of debate, 33,34 the Her2 kinase domain has been suggested to be catalytically active only if it is phosphorylated at this regulatory residue. Xu et al 35 reported that autophosphorylation of Y1248 was decreased after mutation of Y877 to phenylalanine and that Y1248 might occur as a result of Y877 phosphorylation.…”