Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity.
Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the activated EGFR to induce cell growth and transformation, we truncated the human EGFR just after residue 1011, removing all three major autophosphorylation sites (DEL1011). Further, a point mutation was introduced at another autophosphorylation site, Tyr-992-->Phe (DEL1011+F992). The wild-type and mutant receptors were stably expressed in a NIH 3T3 variant cell line that expresses an extremely low level of endogenous EGFR and does not grow with EGF. As expected, DEL1011 and DEL1011+F992 were found to be severely impaired in EGF-induced autophosphorylation, due to the deletion of the appropriate target tyrosines. However, mutant receptors still could induce EGF-dependent DNA synthesis, morphological transformation, and anchorage-independent growth, although the extent of these was significantly reduced when compared with wild-type EGFR. EGF-induced tyrosine phosphorylation of Ras-GTPase activating protein-associated protein p62 and phospholipase C gamma 1 was dramatically reduced in the cells expressing DEL1011 and DEL1011+F992. On the other hand, tyrosine phosphorylation of Shc, complex formation of Shc-Grb2/Ash, and activation of microtubule-associated protein kinase were still fully induced upon EGF stimulation without binding of Shc or Grb2/Ash to the mutant receptor. Thus, tyrosine phosphorylation of Shc may play a crucial role for activating Ras and generating mitotic signals by the activated EGFR mutant.
Gotoh
N
N
Department of Genetics, University of Tokyo, Japan.
Tojo
A
A
Muroya
K
K
Hashimoto
Y
Y
Hattori
S
S
Nakamura
S
S
Takenawa
T
T
Yazaki
Y
Y
Shibuya
M
M
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004261:DNA Replication; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D019204:GTP-Binding Proteins / Q000378:metabolism; D051379:Mice; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D019000:Phosphotyrosine; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D011506:Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins