PLC-GAMMA-1 SRC HOMOLOGY DOMAIN INDUCES MITOGENESIS IN QUIESCENT NIH 3T3 FIBROBLASTS

Previously, we demonstrated that microinjection of phosphoinositide-sp ecific phospholipase C gamma l (PLC gamma l) and lipase-defective muta nts of PLC gamma l induced G(o) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that reg ions other than the catalytic domain of PLCyl may be responsible for i nducing mitogenesis. To test other regions of PLC gamma l for DNA synt hesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial ex pression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA syn thesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial r esponse that was restored to full activity if they were co-injected. T he PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstr ating that non-catalytic structural domains of PLC gamma l have pronou nced effects on mitogenic signaling pathways. (C) 1996 Academic Press, Inc.