cAMP-dependent protein kinase of Manduca sexta phosphorylates but does not activate the fat body triglyceride lipase.
cAMP-dependent-protein kinase (PKA) is a central player of the adipokinetic signal that controls the mobilization of stored lipids in the fat body. Previous studies showed that adipokinetic hormone (AKH) rapidly activates PKA from the fat body of Manduca sexta (Arrese et al. (J. Lipid. Res. 40(3): 556)). As a part of our investigation on lipolysis in insects, here we report the purification and characterization of the catalytic subunit of PKA from the fat body of M. sexta and its role in the direct activation of the TG lipase in vitro. PKA was purified to apparent homogeneity and the identity of the protein was confirmed by MALDI-TOF and Western blot analysis. The enzyme showed a high affinity for Mg-ATP (Km = 39 microM) and Kemptide (Km = 31 microM) and was strongly inhibited by the PKA specific inhibitors PKI 5-24 and H89. Manduca sexta PKA only recognized serine residues as phosphate acceptor; theronine or tyrosine containing peptides were not phosphorylated. Purified fat body TG-lipase proved to be a good substrate of the purified kinase. However, phosphorylation of the lipase did not enhance the lipolytic activity of the enzyme in vitro. These results suggest that, besides lipase phosphorylation, the mechanism of AKH-induced activation of the lipolysis requires the involvement of other proteins and/or signals.
Patel
Rajesh
R
Department of Biochemistry and Molecular Biology, Oklahoma State University, 246 Noble Research Center, Stillwater, OK 74078, USA.
Soulages
Jose L
JL
Wells
Michael A
MA
Arrese
Estela L
EL
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D005216:Fat Body / Q000201:enzymology*; D008049:Lipase / Q000378:metabolism*; D050356:Lipid Metabolism; D018402:Manduca / Q000201:enzymology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D017868:Cyclic AMP-Dependent Protein Kinases; D008049:Lipase