Phosphorylation of phospholipase C-delta 1 regulates its enzymatic activity

Phosphorylation of phospholipase C-delta(1) (PLC-delta(1)) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC-delta(1) most potently. It was also demonstrated that PLC-delta(1) directly bound PKC-alpha via its pleckstrin homology (PH) domain. Using deletion mutants of PLC-delta(1) and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC-delta(1). In vitro phosphorylation of PLC-delta(1) by PKC stimulated [(3)H]PtdIns(4,5)P(2) hydrolyzing activity and [(3)H]Ins(1,4,5)P(3)-binding of the PLC-delta(1). On the other hand, endogenous PLC-delta(1) was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, we determined that Thr209 of PLC-delta(1) is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC-delta(1) was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC-alpha reduced serine phosphorylation of PLC-delta(1) detected by an anti-phosphoserine antibody and PLC-delta(1)-dependent basal production of inositol phosphates in NIH-3T3 cells, suggesting PKC-alpha activates phosphatase or inactivates another kinase involved in PLC-delta(1) serine phosphorylation to modulate the PLC-delta(1) activity in vivo. Taken together, these results suggest that PLC-delta(1) has multiple phosphorylation sites and phosphorylation status of PLC-delta(1) regulates its activity positively or negatively depends on the phosphorylation sites.