Phosphorylation enhances mitochondrial targeting of GSTA4-4 through increased affinity for binding to cytoplasmic Hsp70.
Recently we showed that three different isoforms of cytosolic glutathione S-transferases (GST), including GSTA4-4, are also localized in the mitochondrial compartment. In this study, we have investigated the mechanism of mouse GSTA4-4 targeting to mitochondria, using a combination of in vitro mitochondrial import assay and in vivo targeting in COS cells transfected with cDNA. Our results show that the mitochondrial GSTA4-4 is more heavily phosphorylated compared with its cytosolic counterpart. Protein kinase activators (cAMP, forskolin, or phorbol-12-myristate-13-acetate) markedly increased GSTA4-4 targeting to mitochondria, whereas kinase inhibitors caused its retention in the cytosol. Immunoinhibition and immunodepletion studies showed that the Hsp70 chaperone is required for the efficient translation of GSTA4-4 as well as its translocation to mitochondria. Co-immunoprecipitation studies showed that kinase inhibitors attenuate the affinity of GSTA4-4 for cytoplasmic Hsp70 suggesting the importance of phosphorylation for binding to the chaperone. Mutational analysis show that the putative mitochondrial targeting signal resides within the C-terminal 20 amino acid residues of the protein and that the targeting signal requires activation by phosphorylation at the C-terminal-most protein kinase A (PKA) site at Ser-189 or protein kinase C (PKC) site at Thr-193. We demonstrate for the first time that PKA and PKC modulate the cytoplasmic and mitochondrial pools of GSTA4-4.
Robin
Marie-Anne
MA
Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Prabu
Subbuswamy K
SK
Raza
Haider
H
Anandatheerthavarada
Hindupur K
HK
Avadhani
Narayan G
NG
D000595:Amino Acid Sequence; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D019556:COS Cells; D003062:Codon; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015870:Gene Expression; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007163:Immunosorbent Techniques; D008564:Membrane Potentials; D051379:Mice; D008930:Mitochondria, Liver / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis / Q000235:genetics; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D003062:Codon; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D005576:Colforsin; D003994:Bucladesine; D013762:Tetrahydrofolate Dehydrogenase; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D014357:Trypsin