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

Abstract

The charge isomers of bovine brain PI-TP␣ (i.e. PI-TP␣I containing a phosphatidylinositol (PI) molecule and PI-TP␣II containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the doublereciprocal plot, it was estimated that the V max values for PI-TP␣I 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 M. Phosphorylation of charge isomers of recombinant mouse PI-TP␣ confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo 32 P-labeled PI-TP␣s showed that serine was the major site of phosphorylation. Degradation of 32 P-labeled PI-TP␣ 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-TP␣. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TP␣ to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TP␣ expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TP␣(S166A) and Myc-tagged PI-TP␣(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-TP␣ is linked to the degradation of PI.

Coordinated Lipid Transfer between the Endoplasmic Reticulum and the Golgi Complex Requires the VAP Proteins and Is Essential for Golgi-mediated Transport

“…Phosphorylation of Nir2 at its PI-transfer domain by DAG-activated Golgi localized PKCs, could also provide a mechanism for negatively regulating DAG levels in the Golgi. Previous studies on PI-transfer proteins have shown that phosphorylation of highly conserved residues affects their PI/PC-exchange activity (van Tiel et al, 2000;Morgan et al, 2004). Thus, it could be that similar phosphorylation in the PI-transfer domain of Nir2 affects its PI-and/or-PCtransfer activity, thereby inhibiting DAG production and/or accelerating its consumption.…”

Phosphatidylinositol transfer proteins: From closed for transport to open for exchange

“…More specifically, Snoek et al (2004) have proposed that interaction of PI-TPa with the membrane may induce a slight reorientation of helix F. Since Ser166 is located at a distance of about 6 Å above the axis of helix F, this reorientation could trigger a slight conformational change leading to solvent exposure of the g-OH function of Ser166. Based on a kinetic analysis it was observed that the PCcontaining form of PI-TPa was phosphorylated at a faster rate than the PI-containing form (van Tiel et al, 2000). In line with this, the mutant PI-TPa(T59A), which is unable to bind PI, was a better substrate for PKC than the wtPI-TPa .…”

“…PI-TPa and PI-TPb are substrates for protein kinase C (van Tiel et al, 2000(van Tiel et al, , 2002. Phosphoamino acid analysis of in vitro 32 P-labeled PI-TPs demonstrated that both isoforms were exclusively phosphorylated on a serine residue.…”

“…4B). Despite this distance Ser166 is essential for phospholipid transfer activity as the mutant PI-TPas (S166A, S166D, S166E) fail to transfer PI and PC between membranes (Van Tiel et al, 2000;Morgan et al, 2004). Since the phosphomimetic mutants (S166D, S166E) fail to express transfer activity we may assume that phosphorylated PI-TPa is inactive as well.…”

“…Receptor-mediated activation of phospholipase C resulting in diglyceride production and subsequent PKC activation at the plasma membrane has been proposed to trigger the phosphorylation of PI-TPa (Van Tiel et al, 2000;Morgan et al, 2004). If so, then it appears that this phosphorylated form can relocalize from the plasma membrane to perinuclear Golgi structures (Van Tiel et al, 2000). In contrast to Myc-tagged wtPI-TPa, Myc-tagged mutants (S166A, S166D) expressed in NIH3T3 cells did not relocalize to the Golgi structures upon PMA stimulation, strongly suggesting that phosphorylation of Ser166 is required for relocalization.…”