Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A.
Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
Liauw
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Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Steinberg
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RA
D000818:Animals; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D051379:Mice; D019319:Okadaic Acid; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D013912:Threonine / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D019319:Okadaic Acid; D013912:Threonine; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2