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Functional effects of protein kinases and peroxynitrite on cardiac carnitine palmitoyltransferase-1 in isolated mitochondria

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Abstract

We have previously shown that metoprolol can inhibit carnitine palmitoyltransferase-1 catalytic activity and decrease its malonyl CoA sensitivity within 30 min, suggesting the importance of a covalent modification. The aim of this study was to characterize the effects of PTMs on CPT-1 in the heart. Mitochondria were isolated from the hearts of male Wistar rats and incubated with kinases of interest (protein kinase A, CAMK-II, p38 MAPK, Akt) or with peroxynitrite and sodium nitroprusside. PKA decreased CPT-1 malonyl CoA sensitivity, associated with phosphorylation of CPT-1A, whereas CAMK-II increased malonyl CoA sensitivity by phosphorylating CPT-1B. p38 bound to CPT-1B and stimulated CPT-1 activity. The association of CPT-1 with these kinases and their scaffolding proteins was confirmed in co-localization studies. Peroxynitrite and sodium nitroprusside reversibly stimulated CPT-1 activity, and the change in CPT-1B activity was most consistently associated with glutathiolation of CPT-1B. These studies have identified a new regulatory system of kinases, scaffolding proteins and thiol redox chemistry which can control cardiac CPT-1 in vitro.

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Acknowledgments

We thank Dr. Suzanne Perry, Dr. Shouming He, and Ms. Kyung-Mee Moon at the Proteomics Core Facility, University of British Columbia for carrying out the mass spectrometry analyses. We also thank Dr. Leonard Foster (Department of Biochemistry and Physiology, UBC) for his valuable advice on the mass spectrometry studies. We thank Amrit Samra (iCapture centre, St. Paul’s Hospital, Vancouver) for the preparation of heart cryosections, and Mrs. Hannah Parsons for assistance with preliminary experiments. This work was supported by the Canadian Institutes of Health Research.

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Authors and Affiliations

  1. Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, Canada

    Vijay Sharma, Amie So & John H. McNeill

  2. UBC James Hogg Research Centre, Providence Heart and Lung Institute, The University of British Columbia, 1081 Burrard Street, Vancouver, BC, V6Z 1Y6, Canada

    Thomas Abraham & Michael F. Allard

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  1. Vijay Sharma
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  3. Amie So
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  4. Michael F. Allard
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Correspondence to John H. McNeill.

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11010_2009_303_MOESM1_ESM.jpg

Fig. 1: Overview of experimental design for experiments investigating CPT-1 phosphorylation. Half of each mitochondrial isolate was incubated with the kinase of interest, and the other half received vehicle; each isolate therefore acted as its own control. At the end of the incubation, the isolate was either used for determination of CPT-1 activity, or for co-immunoprecipitation studies. (JPG 47 kb)

11010_2009_303_MOESM2_ESM.ppt

Fig. 2: positive and negative controls for immunoprecipitation experiments. The cytosolic fraction from the mitochondrial isolates was used as a negative control for immunoprecipitations involving CPT-1. Homogenisation buffer containing the purified kinase of interest (in the same concentration used in the kinase incubations) was used as the negative control for the following samples: IP: AKAP-149 blot: PKA, IP: CAMK-II blot: CAMK-II and IP: JIP-2 blot: p38 MAPK. Mitochondrial isolates were used as positive controls. For immunoprecipitations involving nitrosocysteine, glutathione and nitrotyrosine antibodies, the positive controls were mitochondrial isolates pre-incubated with 500 μM peroxynitrite. − negative control, + positive control. (PPT 248 kb)

11010_2009_303_MOESM3_ESM.jpg

Fig. 3: Flow diagram summarizing the results from Fig. 6. Peroxynitrite and sodium nitroprusside induced several modifications simultaneously, which meant that the interpretation of our results was not straightforward. This figure summarises the process of elimination we used to deduce that stimulation of CPT-1 is due to glutathiolation of CPT-1B. (JPG 119 kb)

11010_2009_303_MOESM4_ESM.jpg

Fig. 4: Proposed model of the actions of PKA and CAMK-II. Left panel: Exogenously applied PKA phosphorylates CPT-1A and is then captured by its scaffolding protein. Phosphorylation produces a conformational change tightening the interaction between AKAP-149 and CPT-1A. As a result, malonyl CoA is denied access to its binding site, and the sensitivity of CPT-1 to malonyl CoA is reduced. Note that CPT-1 and AKAP-149 are both anchored in the mitochondrial membrane. Right panel: Exogenously applied CAMK-II phosphorylates CPT-1B and is then captured by its scaffolding protein α-actinin. Phosphorylation produces a conformational change, loosening the interaction between α-actinin and CPT-1B. As a result, malonyl CoA has improved access to its binding site and the sensitivity of CPT-1 to malonyl CoA is increased. Note that CPT-1B is anchored to the mitochondrial membrane whereas α-actinin is anchored to the cytoskeleton. (JPG 55 kb)

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Sharma, V., Abraham, T., So, A. et al. Functional effects of protein kinases and peroxynitrite on cardiac carnitine palmitoyltransferase-1 in isolated mitochondria. Mol Cell Biochem 337, 223–237 (2010). https://doi.org/10.1007/s11010-009-0303-2

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  • DOI: https://doi.org/10.1007/s11010-009-0303-2

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