TY - JOUR T1 - Regulation of Choline Transporter Surface Expression and Phosphorylation by Protein Kinase C and Protein Phosphatase 1/2A JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 536 LP - 545 DO - 10.1124/jpet.104.066795 VL - 310 IS - 2 AU - Jeremiah Gates, Jr. AU - Shawn M. Ferguson AU - Randy D. Blakely AU - Subbu Apparsundaram Y1 - 2004/08/01 UR - http://jpet.aspetjournals.org/content/310/2/536.abstract N2 - The Na+/Cl–-dependent, hemicholinium-3-sensitive choline transporter (CHT) provides choline for acetylcholine biosynthesis. Recent studies show that CHT contains canonical protein kinase C (PKC) serine and threonine residues. We examined the ability of PKC and serine/threonine protein phosphatase 1/2A (PP1/PP2A) to regulate CHT function, surface expression, and phosphorylation. In mouse crude striatal and hippocampal synaptosomes, PKC activators β-phorbol 12-myristate 13-acetate (β-PMA) and β-phorbol 12,13-dibutyrate produced time- and concentration-dependent reductions in CHT function. PP1/PP2A inhibitors okadaic acid (OKA) and calyculin A (CL-A) produced a time- and concentration-dependent decrease in CHT function. However, tautomycin (PP1 inhibitor) and cyclosporin A (PP2B inhibitor) failed to alter CHT-mediated choline uptake. Choline transport kinetic studies following β-PMA, OKA, and CL-A treatment revealed a reduction in the maximal choline transport velocity (Vmax) with no change in Km for choline. These modulators also produced no change in the total levels of CHT protein in the crude hippocampal and striatal synaptosomes; however, surface biotinylation studies using the membrane-impermeant N-hydroxysuccinimide-biotin in crude synaptosomes following treatment with β-PMA, OKA, and CL-A indicate significant reductions of CHT levels in biotinylated fractions. Pretreatment with OKA alone, but not β-PMA, significantly augmented the phosphorylation level of CHT proteins. Our findings suggest that neuronal PKC and PP1/PP2A activity may establish the level of function and surface expression of CHT. These studies also provide the first evidence that CHT is a phosphoprotein and that the basal PP1/PP2A activity may have a dominant role in controlling the levels of CHT phosphorylation. The American Society for Pharmacology and Experimental Therapeutics ER -