(1) The mechanism by which PDGF and EGF activate phospholipase C was studied. Stimulation with the growth factors promotes the association of PLC-gamma with the growth factor receptors and increase of tyrosine and semine phosphorylation of PLC-gamma. We identified 4 tyrosine phosphorylation sites in PLC-gamma. The tyrosine phosphorylation did not affect the catalytic activity of PLC-gamma in vitro. We proposed, therefore, that the phosphorylation of PLC-gamma by EGF receptor kinase alters its interaction with putative inhibitory protein and leads to its activation. (2) Treatment of NIH 3T3 cells with EGF or PDGF induced a translocation of PLC-gamma from cytosol to membrane. (3) Although the nerve growth factor (NGF) receptor is not a protein kinase, NGF induces an increase in tyrosine and serine phosphorylation. This result suggests the possibility that the NGF-dependent increase in phosphoinositide hydrolysis in PC12 cells is due to selective phosphorylation of PLC-gamma by serine and tyrosine protein kinase coupled to the NGF receptor. (4) Activation of protein kinase C attenuates the receptor-coupled PLC activity and provides a negative feedback signal to limit the magnitude and duration of receptor signalling. We identified PLC-beta and EGF receptor as the target of protein kinase C. (5) A rapid method for purification of inositol (1,4,5)trisphosphate 3-kinase (IP3K) from rat brain was developed. Inclusion of calpain inhibitor prevented the progressive degradation of the 53-kDa protein to several catalytically active fragments. Using the nonproteolyzed 53-kDa enzyme, we showed that cAMP-dependent protein kinase and protein kinase C phosphorylate, and thereby modulate the activity of IP3K. (6) Monoclonal antibodies to IP3K were prepared. Using these antibodies, we isolated a cDNA clone that encodes IP3K from a rat brain cDNA expression library. This clone had an open reading frame that would direct the synthesis of a protein of 449 amino acids. The putative protein revealed a potential calmodulin-binding site and 6 regions with amino acid compositions common to proteins that are susceptible to calpain.
