Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins*

Amplification of the type I receptor tyrosine kinase ErbB-2 (HER2/Neu) is observed in 20–30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Tyr1144, Tyr1201, Tyr1226/1227, or Tyr1253) of the five known Neu/ErbB-2 autophosphorylation sites can independently mediate transforming signals. The transforming potential of at least two of these autophosphorylation sites (Tyr1144and Tyr1226/1227) has been further correlated with their ability to associate with Grb2 and Shc adapter proteins, respectively. To confirm the specificity of these interactions, we have created a series of second site mutants in these phosphorylation sites. The results showed that Grb2 recruitment to site 1144 is absolutely required for transforming signal from this autophosphorylation site, whereas association of Shc-mediated transformation is dependent on conservation of the NPXY motif spanning Tyr1227. A stretch of amino acid identity around tyrosines 1201 (ENPEYLTP)and 1253 (ENPEYLDL) exists, and mutation of key residues within this motif reveals distinct requirements for an intact protein tyrosine-binding protein (NPXY). We show that DOK-R, a protein tyrosine-binding site-containing protein implicated in Ras signaling, interacts with Neu/ErbB-2 at Tyr1253 as do two unidentified proteins, p150 and p34, the latter correlating with transformation. Together these data argue that ErbB-2/Neu is capable of mediating transformation through distinct effector pathways.

[1]  M. Moran,et al.  Grb2 and Shc Adapter Proteins Play Distinct Roles in Neu (ErbB-2)-Induced Mammary Tumorigenesis: Implications for Human Breast Cancer , 2001, Molecular and Cellular Biology.

[2]  H. Hayasawa,et al.  p56dok‐2 as a cytokine‐inducible inhibitor of cell proliferation and signal transduction , 2000 .

[3]  Monilola A. Olayioye,et al.  The ErbB signaling network: receptor heterodimerization in development and cancer , 2000, The EMBO journal.

[4]  W. Muller,et al.  Signal transduction in mammary tumorigenesis: a transgenic perspective , 2000, Oncogene.

[5]  D. Baltimore,et al.  Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling. , 2000, Genes & development.

[6]  D. Dumont,et al.  The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R , 1998, Oncogene.

[7]  W. Paul,et al.  FRIP, a hematopoietic cell-specific rasGAP-interacting protein phosphorylated in response to cytokine stimulation. , 1998, Immunity.

[8]  S. Bull,et al.  neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto Breast Cancer Study Group. , 1998, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[9]  M. Moran,et al.  Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation , 1997, Molecular and cellular biology.

[10]  H. Band,et al.  Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity , 1997, Molecular and cellular biology.

[11]  M. Shibuya,et al.  Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation , 1997, Molecular and cellular biology.

[12]  S. Harrison,et al.  Peptide–Surface Association: The Case of PDZ and PTB Domains , 1996, Cell.

[13]  M. F. White,et al.  Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Tony Pawson,et al.  Protein modules and signalling networks , 1995, Nature.

[15]  N. Hynes,et al.  The biology of erbB-2/neu/HER-2 and its role in cancer. , 1994, Biochimica et biophysica acta.

[16]  B. Margolis,et al.  A region in Shc distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors. , 1994, The Journal of biological chemistry.

[17]  M. Maa,et al.  A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma , 1994, Molecular and cellular biology.

[18]  Y. Yarden,et al.  A single autophosphorylation site confers oncogenicity to the Neu/ErbB‐2 receptor and enables coupling to the MAP kinase pathway. , 1994, The EMBO journal.

[19]  T Pawson,et al.  Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav , 1994, Molecular and cellular biology.

[20]  T. Pawson,et al.  Shc products are substrates of erbB-2 kinase. , 1993, Oncogene.

[21]  D. Ron,et al.  pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins. , 1992, BioTechniques.

[22]  A. Ullrich,et al.  SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high‐affinity binding site for SH2 domains of phospholipase C gamma. , 1992, The EMBO journal.

[23]  H. Land,et al.  A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells. , 1990, Nucleic acids research.

[24]  M. Moran,et al.  Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases , 1990, Nature.

[25]  W Godolphin,et al.  Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. , 1989, Science.

[26]  W. McGuire,et al.  Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. , 1987, Science.