Phosphorylation of rat liver mitochondrial carnitine palmitoyltransferase-I: effect on the kinetic properties of the enzyme.
Hepatic carnitine palmitoyltransferase-I (CPT-IL) isolated from mitochondrial outer membranes obtained in the presence of protein phosphatase inhibitors is readily recognized by phosphoamino acid antibodies. Mass spectrometric analysis of CPT-IL tryptic digests revealed the presence of three phosphopeptides including one with a protein kinase CKII (CKII) consensus site. Incubation of dephosphorylated outer membranes with protein kinases and [gamma-32P]ATP resulted in radiolabeling of CPT-I only by CKII. Using mass spectrometry, only one region of phosphorylation was detected in CPT-I isolated from CKII-treated mitochondria. The sequence of the peptide and position of phosphorylated amino acids have been determined unequivocally as FpSSPETDpSHRFGK (residues 740-752). Furthermore, incubation of dephosphorylated outer membranes with CKII and unlabeled ATP led to increased catalytic activity and rendered malonyl-CoA inhibition of CPT-I from competitive to uncompetitive. These observations identify a new mechanism for regulation of hepatic CPT-I by phosphorylation.
Kerner
Janos
J
Department of Nutrition, Biochemistry, and Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Distler
Anne M
AM
Minkler
Paul
P
Parland
William
W
Peterman
Scott M
SM
Hoppel
Charles L
CL
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000502:physiology*; D047390:Casein Kinase II; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007477:Ions; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008316:Malonyl Coenzyme A / Q000737:chemistry; D013058:Mass Spectrometry; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D007477:Ions; D010455:Peptides; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D014357:Trypsin