Phosphorylation of sterol regulatory element binding protein-1a by protein kinase A (PKA) regulates transcriptional activity.
The counter-regulatory hormone glucagon inhibits lipogenesis via downregulation of sterol regulatory element binding protein 1 (SREBP-1). The effect of glucagon is mediated via protein kinase A (PKA). To determine if SREBP-1 is a direct phosphorylation target of PKA, we conducted mass spectrometry analysis of recombinant n-terminal SREBP-1a following PKA treatment in vitro. This analysis identified serines 331/332 as bona-fide phosphorylation targets of PKA. To determine the functional consequences of phosphorylation at these sites, we constructed mammalian expression vector for both nSREBP-1a and 1c isoforms in which the candidate PKA phosphorylation sites were mutated to active phosphomimetic or non-phosphorylatable amino acids. The transcriptional activity of SREBP was reduced by the phosphomimetic mutation of S332 of nSREBP-1a and the corresponding serine (S308) of nSREBP-1c. This site is a strong candidate for mediating the negative regulatory effect of glucagon on SREBP-1 and lipogenesis.
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA.
Qingming
Q
Dong
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA.
Francesco
F
Giorgianni
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA; Research Service, Veteran's Affairs Medical Center, Memphis, TN, USA.
Xiong
X
Deng
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA.
Sarka
S
Beranova-Giorgianni
Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, USA.
Dave
D
Bridges
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA.
Edwards A
EA
Park
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA; Research Service, Veteran's Affairs Medical Center, Memphis, TN, USA.
Rajendra
R
Raghow
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN, USA; Research Service, Veteran's Affairs Medical Center, Memphis, TN, USA. Electronic address: melam@uthsc.edu.
Marshall B
MB
Elam
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005934:Glucagon / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D010766:Phosphorylation; D016415:Sequence Alignment; D051780:Sterol Regulatory Element Binding Protein 1 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D051780:Sterol Regulatory Element Binding Protein 1; D005934:Glucagon; D017868:Cyclic AMP-Dependent Protein Kinases
Immobilized metal affinity chromatography (IMAC); Mass spectrometry (MS); Phosphorylation; Protein kinase A (PKA); Site-directed mutagenesis; Sterol regulatory element binding protein (SREBP)