The role of the Shc phosphotyrosine interaction/phosphotyrosine binding domain and tyrosine phosphorylation sites in polyoma middle T antigen-mediated cell transformation.
The phosphotyrosine interaction (PI)/phosphotyrosine binding (PTB) domain of Shc binds specific tyrosine-phosphorylated motifs found on activated growth factor receptors and proteins such as polyoma virus middle T antigen (MT). Phenylalanine 198 (Phe198) has been identified as a crucial residue involved in the interaction of the Shc PI/PTB with phosphopeptides. In NIH 3T3 cells expressing MT, p52 Shc carrying the F198V mutation is weakly phosphorylated and does not bind MT or Grb2. Overexpression of the PI/PTB domain alone as Shc amino acids 1-238 acted in a dominant interfering fashion blocking MT-induced transformation. However, expression of a slightly longer construct, Shc 1-260, which encompasses Tyr239/Tyr240, a novel Shc tyrosine phosphorylation site, did not block transformation. This was found to be due to the ability of Shc 1-260 to become tyrosine-phosphorylated and bind Grb2. Furthermore, full-length Shc in which Tyr239/Tyr240 had been mutated to phenylalanine did not become tyrosine-phosphorylated or bind Grb2 but did inhibit colony formation in soft agar. Conversely, p52 Shc carrying a mutation in the other tyrosine phosphorylation site, Tyr317, became heavily tyrosine-phosphorylated, bound Grb2, and gave rise to colonies in soft agar.
Blaikie
P A
PA
Department of Internal Medicine and Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0650, USA.
Fournier
E
E
Dilworth
S M
SM
Birnbaum
D
D
Borg
J P
JP
Margolis
B
B
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D001665:Binding Sites; D002471:Cell Transformation, Neoplastic; D051380:GRB2 Adaptor Protein; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000952:Antigens, Polyomavirus Transforming; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D014443:Tyrosine