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 subu nit 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 b een assumed to be metabolically stable. We now show that this phosphat e can be removed from C subunit by a protein phosphatase activity extr acted from S49 mouse lymphoma cells or by purified protein phosphatase -2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exch ange chromatography, inhibitor sensitivity, and relative activity agai nst glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffectiv e 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 phospha te-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 extrac ts 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.