PHOSPHOLIPASE C-GAMMA-1 CAN INDUCE DNA-SYNTHESIS BY A MECHANISM INDEPENDENT OF ITS LIPASE ACTIVITY

Inositol phospholipid-specific phospholipase C (PLC) is involved in se veral signaling pathways leading to cellular growth and differentiatio n. Our previous studies reported the induction of DNA synthesis in qui escent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal ant ibodies to PLC-gamma 1. In the course of our investigation of anti-PLC -gamma 1 monoclonal antibodies, we found that each antibody exerts dif ferent inhibitory effects on the phosphatidylinositol-hydrolyzing acti vity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the mi croinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC -gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzym es were expressed using a vaccinia expression system. The mutant enzym es were purified and microinjected into quiescent NIH 3T3 cells to eva luate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activit y was inhibited by an antibody (alpha E 8-4) that does not significant ly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA s ynthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. Thes e results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells.