We previously reported that the activity of γ-glutamylcysteine synthetase (GCS; EC 18.104.22.168), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited ∼20–40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260–269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg2+-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM 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 phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its Km. Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.
Research Article|November 15 1996
Regulation of γ-glutamylcysteine synthetase by protein phosphorylation
Shelly C LU
Shelly C LU
1Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California School of Medicine, 1333 San Pablo St., Los Angeles, CA 90033, U.S.A.
*To whom correspondence should be addressed.
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Wei-Min SUN, Zong-Zhi HUANG, Shelly C LU; Regulation of γ-glutamylcysteine synthetase by protein phosphorylation. Biochem J 15 November 1996; 320 (1): 321–328. doi: https://doi.org/10.1042/bj3200321
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