Phosphorylation of rat liver mitochondrial glycerol-3-phosphate acyltransferase by casein kinase 2.
We have previously shown rat liver mitochondrial glycerol-3-phosphate acyltransferase (mtGAT), which catalyzes the first step in de novo glycerolipid biosynthesis, is stimulated by casein kinase 2 (CK2) and that a phosphorylated protein of approximately 85 kDa is present in CK2-treated mitochondria. In this paper, we have identified the (32)P-labeled 85-kDa protein as mtGAT. We have also investigated whether the phosphorylation of mtGAT is because of CK2. Mitochondria were treated with CK2 and [gamma-(32)P]GTP as the phosphate donor. Autoradiography, Western blot, and immunoprecipitation results showed mtGAT was phosphorylated by CK2. Next, we incubated mitochondria with CK2 and either ATP or GTP, in the presence of heparin, a known inhibitor of CK2. Heparin inhibited CK2-induced stimulation of mtGAT activity; this inhibition resulted in decreased (32)P-labeling of mtGAT. Additionally, mitochondria were treated with CK2 and [gamma-(32)P]ATP in the presence of staurosporine (a serine/threonine protein kinase inhibitor), genistein (a tyrosine kinase inhibitor), and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB, a CK2 inhibitor). Only DRB, the CK2 inhibitor, greatly reduced the amount of (32)P-incorporation into mtGAT by CK2. Finally, isolated mitochondrial outer membrane was incubated with cytosol in the presence of [gamma-(32)P]GTP; (32)P-labeled mtGAT was detected. Collectively, these data suggest that CK2 phosphorylates mtGAT. The impact of our results in the regulation of mtGAT and other anabolic processes is discussed.
Onorato
Thomas M
TM
Department of Biological Sciences, St. John's University, Queens, New York 11439, USA.
Chakraborty
Sanjoy
S
Haldar
Dipak
D
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites / Q000235:genetics; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D010761:Phosphorus Radioisotopes; D004004:Dichlororibofuranosylbenzimidazole; D006493:Heparin; D019833:Genistein; D005992:Glycerol-3-Phosphate O-Acyltransferase; D047390:Casein Kinase II; D019311:Staurosporine