Kerner Janos, Distler Anne M, Minkler Paul, Parland William, Peterman Scott M, Hoppel Charles L
Department of Nutrition, Biochemistry, and Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2004 Sep 24;279(39):41104-13. doi: 10.1074/jbc.M406570200. Epub 2004 Jul 9.
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.
从存在蛋白磷酸酶抑制剂的情况下获得的线粒体外膜中分离出的肝肉碱棕榈酰转移酶-I（CPT-IL）很容易被磷酸氨基酸抗体识别。对CPT-IL胰蛋白酶消化产物进行质谱分析，发现存在三种磷酸肽，其中一种具有蛋白激酶CKII（CKII）的共有位点。用蛋白激酶和[γ-32P]ATP对去磷酸化的外膜进行孵育，结果只有CKII能使CPT-I放射性标记。通过质谱分析，在从经CKII处理的线粒体中分离出的CPT-I中仅检测到一个磷酸化区域。已明确确定磷酸化氨基酸的肽序列和位置为FpSSPETDpSHRFGK（第740 - 752位氨基酸）。此外，用CKII和未标记的ATP对去磷酸化的外膜进行孵育会导致催化活性增加，并使丙二酰辅酶A对CPT-I的抑制作用从竞争性转变为非竞争性。这些观察结果确定了一种通过磷酸化调节肝CPT-I的新机制。
