title: The human glutathione S-transferase P1 protein is phosphorylated and its metabolic function enhanced by the Ser/Thr protein kinases, cAMP-dependent protein kinase and protein kinase C, in glioblastoma cells.
abstract: We report here that the human glutathione S-transferase P1 (GSTP1) protein, involved in phase II metabolism of many carcinogens and anticancer agents and in the regulation of c-Jun NH(2)-terminal kinase-mediated cell signaling, undergoes phosphorylation by the Ser/Thr protein kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), resulting in a significant enhancement of its metabolic activity. GSTP1 phosphorylation by PKA was glutathione (GSH)-dependent, whereas phosphorylation by PKC did not require but was significantly enhanced by GSH. In the presence of GSH, the stoichiometry of phosphorylation was 0.4 +/- 0.03 and 0.53 +/- 0.02 mol incorporated phosphate per mole of dimeric GSTP1 protein. The GSTP1 protein was phosphorylated, in the presence of GSH, by eight different PKC isoforms (alpha, betaIota, betaIotaIota, delta, epsilon, gamma, eta, and zeta), belonging to the three major PKC subclasses, albeit with various efficiencies. The catalytic efficiency, k(cat)/K(m), of the phosphorylated GSTP1 was more than double that of the unphosphorylated protein. In MGR3 human glioblastoma cells, PKA and PKC activation resulted in a significant increase in the level of phosphorylation of the GSTP1 protein and was accompanied by a 2.1- and 2.7-fold increase, respectively, in specific GSTP1 activity in the cells. Peptide phosphorylation analyses and both phosphorylation and enzyme kinetic studies with GSTP1 proteins mutated at candidate amino acid residues established Ser-42 and Ser-184 as putative phospho-acceptor residues for both kinases in the GSTP1 protein. Together, these findings show PKA- and PKC-dependent phosphorylation as a significant post-translational mechanism of regulation of GSTP1 function. The GSH-dependence of the phosphorylation suggests that under high intracellular GSH conditions, such as is present in most drug-resistant tumors, the GSTP1 protein will exist in a hyper-phosphorylated and enzymatically more active state. In normal cells, the functional activation of the GSTP1 protein by PKA- and PKC-dependent phosphorylation could represent a potentially important mechanism of cellular protection, whereas in tumors, increased phase II metabolism of anticancer drugs by the more active phosphorylated GSTP1 protein could contribute to the drug resistance and therapeutic failure frequently associated with increased activities of these Ser/Thr kinases.
authors.affiliation: Department of Surgery and the Comprehensive Cancer Center, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Hui-Wen
authors.initials: HW
authors.lastname: Lo
authors.forename: Gamil R
authors.initials: GR
authors.lastname: Antoun
authors.forename: Francis
authors.initials: F
authors.lastname: Ali-Osman
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001254:Astrocytoma / Q000201:enzymology*; D001665:Binding Sites; D045744:Cell Line, Tumor; D002474:Cell-Free System; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005909:Glioblastoma / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D000217:Acyltransferases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C043403:fatty acyl ethyl ester synthase