The protein kinase C-dependent phosphorylation of serine 166 is controlled by the phospholipid species bound to the phosphatidylinositol transfer protein alpha.
The charge isomers of bovine brain PI-TPalpha (i.e. PI-TPalphaI containing a phosphatidylinositol (PI) molecule and PI-TPalphaII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V(max) values for PI-TPalphaI and II were 2.0 and 6.0 nmol/min, respectively; the K(m) values for both charge isomers were about equal, i.e. 0.7 micrometer. Phosphorylation of charge isomers of recombinant mouse PI-TPalpha confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo (32)P-labeled PI-TPalphas showed that serine was the major site of phosphorylation. Degradation of (32)P-labeled PI-TPalpha by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one (32)P-labeled peptide (amino acids 104-190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPalpha. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPalpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPalpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPalpha(S166A) and Myc-tagged PI-TPalpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPalpha is linked to the degradation of PI.
van Tiel
C M
CM
Center for Biomembranes and Lipid Enzymology, Department of Lipid Biochemistry, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Westerman
J
J
Paasman
M
M
Wirtz
K W
KW
Snoek
G T
GT
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D010449:Peptide Mapping; D010716:Phosphatidylinositols / Q000378:metabolism; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity
D002352:Carrier Proteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011994:Recombinant Proteins; D012694:Serine; D011493:Protein Kinase C