Overexpression of SH2-SH2-SH3 domain of phospholipase C-γ1 blocks PDGF-induced inositol phosphate generation in NIH 3T3 cells

Src homology (SH) 2 and 3 domains are known to be binding motifs for protein-protein interaction in signaling molecules. Among several PLC isozymes, only PLC-γ contains the SH domain between the X and Y domains, which are known to have catalytic activity. To elucidate the functional roles of the SH2-SH2-SH3 domain of PLC-γ1 in cellular signaling, we constructed a truncated cDNA encoding the SH2-SH2-SH3 domain of PLC-γ1 (p60^SH2/SH3) and expressed it in NIH 3T3 cells. Cells expressing p60^SH2/SH3 did not show any change in cell shape no oncogenesity. Even though in a serum depleted condition, a portion of p60^SH2/SH3 existed as constitutively phosphorylated on its tyrosine residues. Furthermore, cells expressing p60^SH2/SH3 did not respond to PDGF-induced IPs formation whereas vector transfected control cells showed dose-dependent IPs generation upon PDGF stimulation. The tyrosine phosphorylation level of endogenous PLC-γ1 by PDGF, however, was comparable to that of the control cells. On the other hand, IPs accumulation by PLC-β activation occurred to a comparable level. Taken together, p60^SH2/SH3 molecules selectively inhibited the IPs accumulation catalyzed by PLC-γ1. This result suggests that the SH2-SH2-SH3 domain is essential for PLC-γ1-mediated cellular signaling, including its own catalytic activity by protein-protein interaction.