REGULATION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE BY PROTEIN-PHOSPHORYLATION

We previously reported that the activity of gamma-glutamylcysteine syn thetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited similar to 20-40% by agonists of various sign al transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ru iz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now exa mined the possibility that GCS is phosphorylated directly by activatio n of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmoduli n-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunopre cipitating the reaction product with specific rabbit anti-(rat GCS hea vy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg2+-concentration-dependen t manner, with the highest degree of phosphorylation occurring at 20 m M Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1 .17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylat ion was correlated with the degree of loss of GCS activity, and no add itional inhibition occurred when GCS was phosphorylated by all three k inases, suggesting that the kinases phosphorylated the same site(s). P hosphoamino analysis showed that all three kinases phosphorylated seri ne and threonine residues. Two-dimensional phosphopeptide mapping demo nstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phos phorylated one additional peptide. Phosphorylation of GCS decreased it s V-max for cysteine and glutamate without changing its K-m. Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylep hrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonst rated that GCS is phosphorylated and suggest that phosphorylation/deph osphorylation may regulate GCS activity.