Biochemical characterization of two forms of 3-hydroxy-3-methylglutaryl-CoA reductase kinase from cauliflower (Brassica oleracia).
We recently reported the existence of a protein kinase cascade in higher plants, of which the central component is a 3-hydroxy-3-methylglutaryl(HMG-)-CoA reductase kinase functionally related to mammalian AMP-activated protein kinase [MacKintosh, R. W., Davies, S. P., Clarke P. R., Weekes, J., Gillespie, S. G., Gibb, B. J. & Hardie, D. G. (1992) Eur. J. Biochem. 209, 923-931]. We have now purified this protein kinase 9000-fold from cauliflower inflorescences. During the course of this work we noticed a second minor form (form B) which separated from the major form (A) on ion exchange and gel filtration. Both forms phosphorylate the catalytic fragment of mammalian HMG-CoA reductase. Both forms are markedly inactivated by incubation with the reactive ATP analogue p-fluorosulphonylbenzoyl adenosine (FSO2PhCOAdo), and also by mammalian protein phosphatase 2C, indicating that form B, like form A, is activated by phosphorylation. Form A has an apparent native molecular mass of 200 kDa by gel filtration and, after labelling with [14C]FSO2PhCOAdo, of 150 kDa by electrophoresis in non-denaturing gels. The catalytic subunit was identified as a polypeptide of 58 kDa after labelling with [14C]FSO2PhCOAdo. Form B has an apparent native molecular mass of 45 kDa by gel filtration, and was identified as a polypeptide of 45 kDa after labelling with [14C]FSO2PhCOAdo and [gamma-32P]ATP. Using a series of variants of the synthetic peptide substrate, the substrate specificities of the two forms are similar but not identical. Form B does not appear to be a proteolytic fragment of form A, and we therefore propose that it represents a closely related member of the same protein kinase sub-family.
Ball
K L
KL
Biochemistry Department, The University, Dundee, Scotland.
Dale
S
S
Weekes
J
J
Hardie
D G
DG
D055372:AMP-Activated Protein Kinases; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001937:Brassica / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D009842:Oligopeptides; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D006903:Hydroxymethylglutaryl CoA Reductases; D025782:Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D000241:Adenosine