The protein kinase C-dependent phosphorylation of serine 166 is controlledby the phospholipid species bound to the phosphatidylinositol transfer protein alpha

The charge isomers of bovine brain PI-TP alpha (i.e. PI-TP alpha I containi ng a phosphatidylinositol (PI) molecule and PI-TP alpha II containing a pho sphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain p rotein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V-max values for PI-TP alpha I and II were 2.0 an d 6.0 nmol/min, respectively; the K-m values for both charge isomers were a bout equal, i.e. 0.7 mu M. Phosphorylation of charge isomers of recombinant mouse PI-TP alpha confirmed that the PC-containing isomer was the better s ubstrate. Phosphoamino acid analysis of in vitro and in vivo P-32-labeled P I-TP alpha s showed that serine was the major site of phosphorylation, Degr adation of P-32-labeled PI-TP alpha by cyanogen bromide followed by high pr essure liquid chromatography and sequence analysis yielded one P-32-labeled peptide (amino acids 104-190), This peptide contained Ser-148, Ser-152, an d the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 wi th an alanine residue confirmed that indeed this residue was the site of ph osphorylation. This mutation completely abolished PI and PC transfer activi ty. This was also observed when Ser-166 was replaced with Asp, implying tha t this is a key amino acid residue in regulating the function of PI-TP alph a, Stimulation of NIH3T3 fibroblasts by phorbol eater or platelet-derived g rowth factor induced the rapid relocalization of PI-TP alpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidyli nositol levels. This relocalization was also observed for Myc-tagged wtPI-T P alpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tag ged PI-TP alpha(S166A) and Myc-tagged PI-TP alpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequi site for the relocalization to the Golgi, A model is proposed in which the PKC-dependent phosphorylation of PI-TP alpha is linked to the degradation o f PI.