GRB2 and phospholipase C-gamma 1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation.
GRB2, a 25-kDa protein comprising a single SH2 domain flanked by two SH3 domains, has been implicated in linking receptor protein tyrosine kinases (PTKs) to the Ras pathway by interacting with the guanine nucleotide exchange protein SOS. Previous studies have demonstrated that GRB2 directly interacts with Shc, a proto-oncogene product that is tyrosine phosphorylated upon receptor and nonreceptor PTK activation. In this report, we detected low levels of tyrosine phosphorylation of Shc and induced association with GRB2 upon T-cell receptor (TCR) stimulation. Instead, a prominent 36- to 38-kDa tyrosine phosphoprotein (pp36-38) associated with the SH2 domain of GRB2 and formed a stable complex with GRB2/SOS upon TCR stimulation. Cellular fractionation studies showed that whereas both GRB2 and SOS partitioned to the soluble and particulate fractions, pp36-38 was present exclusively in the particulate fraction. This phosphoprotein had the same apparent mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the phosphoprotein that associates with phospholipase C-gamma 1 (PLC-gamma 1). Furthermore, following partial immunodepletion of GRB2 and of the associated pp36-38, there was a significant reduction in the amount of the 36-kDa phosphoprotein associated with PLC-gamma 1, suggesting that a trimeric PLC-gamma 1/pp36-38/GRB2 complex could form. In support of this notion, we have also been able to detect low levels of PLC-gamma 1 in GRB2 immunoprecipitates. We suggest that pp36-38 may be a bridging protein, coupling different signalling molecules to cytoplasmic PTKs regulated by the TCR.
Sieh
M
M
Department of Medicine, Howard Hughes Medical Institute, University of California at San Francisco 94143.
Batzer
A
A
Schlessinger
J
J
Weiss
A
A
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D019000:Phosphotyrosine; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007527:Isoenzymes; C000715470:MAS1 protein, human; D010750:Phosphoproteins; D011506:Proteins; D000090063:Proto-Oncogene Mas; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D010738:Type C Phospholipases