The Protein Kinase C-dependent Phosphorylation of Serine 166 Is Controlled by the Phospholipid Species Bound to the Phosphatidylinositol Transfer Protein α*

The charge isomers of bovine brain PI-TPα (i.e. PI-TPαI containing a phosphatidylinositol (PI) molecule and PI-TPαII 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-TPαI and II were 2.0 and 6.0 nmol/min, respectively; theK m values for both charge isomers were about equal,i.e. 0.7 μm. Phosphorylation of charge isomers of recombinant mouse PI-TPα confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32P-labeled PI-TPαs showed that serine was the major site of phosphorylation. Degradation of 32P-labeled PI-TPα by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one32P-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-TPα. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPα to perinuclear Golgi structures concomitant with a 2–3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPα expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPα(S166A) and Myc-tagged PI-TPα(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-TPα is linked to the degradation of PI.

• Publication year

  2000

• Venue

  Journal of Biological Chemistry

• Publication date

  2000-07-14

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M002203200PMID 10801835
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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