Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits.
The alpha form of the A subunit of human protein phosphatase 2A was expressed in insect cells following infection with a recombinant baculovirus. A alpha was expressed as a soluble protein that comprised approximately 10% of total cellular protein. The expressed A alpha subunit was purified by chromatography on amino-hexyl-Sepharose and Mono Q with a yield of 2 mg/500-ml culture. The recombinant protein had the same apparent molecular mass as the bovine cardiac protein and was devoid of myosin light chain phosphatase activity. Biological activity of expressed A was assessed by assays of complex formation with the catalytic (C) and B subunits, purified from bovine cardiac tissue, and by inhibition of phosphatase activity. Purified A alpha had a high apparent affinity for C (IC50 = 0.10 nM) and bound with a stoichiometry of 1 mol of A/mol of C. Interaction of A alpha with the catalytic subunit caused a maximal inhibition of myosin light chain and phosphorylase phosphatase activities of 50 and 79%, respectively. The AC complex prepared by reconstitution of recombinant A alpha with C had the same electrophoretic mobility in nondenaturing polyacrylamide gels and the same elution volume when chromatographed on a size exclusion column as the native AC complex purified from cardiac muscle. Similar chromatographic profiles were also observed for the heterotrimer reconstituted from recombinant A alpha, purified B and C, and the native bovine cardiac heterotrimeric holoenzyme. Cross-linking of the native enzyme and the reconstituted heterotrimer generated the same pattern of high molecular weight species. Immunological analyses of these complexes demonstrated that distinct cross-linked forms composed of ABC, AC, AB, and BC were obtained. These results suggest that each of the three subunits of protein phosphatase 2A forms direct contacts with both of the others.
Kamibayashi
C
C
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Lickteig
R L
RL
Estes
R
R
Walter
G
G
Mumby
M C
MC
D000818:Animals; D016367:Baculoviridae; D002384:Catalysis; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004247:DNA; D006801:Humans; D009206:Myocardium / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D054648:Protein Phosphatase 2; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D004247:DNA; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2