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Ser649 and Ser650 a...
Ser649 and Ser650 are the major determinants of protein kinase A-mediated activation of human hormone-sensitive lipase against lipid substrates.
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- Krintel, Christian (författare)
- Lund University,Lunds universitet,Molekylär endokrinologi,Forskargrupper vid Lunds universitet,Molecular Endocrinology,Lund University Research Groups
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- Osmark, Peter (författare)
- Lund University,Lunds universitet,Molekylär endokrinologi,Forskargrupper vid Lunds universitet,Molecular Endocrinology,Lund University Research Groups
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Larsen, Martin R (författare)
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- Resjö, Svante (författare)
- Lund University,Lunds universitet,Institutionen för experimentell medicinsk vetenskap,Medicinska fakulteten,Department of Experimental Medical Science,Faculty of Medicine
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- Logan, Derek (författare)
- Lund University,Lunds universitet,Biokemi och Strukturbiologi,Centrum för Molekylär Proteinvetenskap,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biochemistry and Structural Biology,Center for Molecular Protein Science,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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- Holm, Cecilia (författare)
- Lund University,Lunds universitet,Molekylär endokrinologi,Forskargrupper vid Lunds universitet,Molecular Endocrinology,Lund University Research Groups
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(creator_code:org_t)
- 2008-11-19
- 2008
- Engelska.
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:11
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Abstract
Ämnesord
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- BACKGROUND: Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. Its activity is regulated by reversible protein phosphorylation. In rat HSL Ser563, Ser659 and Ser660 have been shown to be phosphorylated by protein kinase A (PKA) in vitro as well as in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we employed site-directed mutagenesis, in vitro phosphorylation and mass spectrometry to show that in vitro phosphorylation of human HSL by PKA occurs primarily on Ser649 and Ser650 (Ser659 and Ser660 in rat HSL). The wild type enzyme and four mutants were expressed in C-terminally His-tagged form in Sf9 insect cells and purified to homogeneity. HSL variants in which Ser552 and/or Ser554 were mutated to Ala or Glu retained both lipolytic and non-lipolytic activity and were phosphorylated by PKA and activated to a similar extent as the wild type enzyme. (32)P-labeling studies revealed that the bulk of the phosphorylation was on the Ser649/Ser650 site, with only a minor phosphorylation of Ser552 and Ser554. MS/MS analysis demonstrated that the peptide containing Ser649 and Ser650 was primarily phosphorylated on Ser650. The mutant lacking all four serines had severely reduced lipolytic activity, but a lesser reduction in non-lipolytic activity, had S(0.5) values for p-nitrophenol butyrate and triolein comparable to those of wild type HSL and was not phosphorylated by PKA. PKA phosphorylation of the wild type enzyme resulted in an increase in both the maximum turnover and S(0,5) using the TO substrate. CONCLUSIONS: Our results demonstrate that PKA activates human HSL against lipid substrates in vitro primarily through phosphorylation of Ser649 and Ser650. In addition the results suggest that Ser649 and Ser650 are located in the vicinity of a lipid binding region and that PKA phosphorylation controls the accessibility of this region.
Ämnesord
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin -- Endokrinologi och diabetes (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine -- Endocrinology and Diabetes (hsv//eng)
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