The amino-terminal Src homology 2 domain of phospholipase C gamma 1 is essential for TCR-induced tyrosine phosphorylation of phospholipase C gamma 1.
TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.
Stoica
B
B
Laboratory of Immunobiology, OTRR, Center for Biologics Evaluation and Research, Bethesda, MD 20892, USA.
DeBell
K E
KE
Graham
L
L
Rellahan
B L
BL
Alava
M A
MA
Laborda
J
J
Bonvini
E
E
D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D002417:Cattle; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D019169:Jurkat Cells; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D018909:src Homology Domains / Q000187:drug effects / Q000235:genetics / Q000276:immunology*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D007527:Isoenzymes; D010750:Phosphoproteins; D011506:Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D001120:Arginine; D005982:Glutathione Transferase; D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma