Abstract
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.
Highlights
Catalytic (C)1 subunit of cAMP-dependent protein kinase when isolated from animal tissues is phosphorylated at two sites, Thr-197 and Ser-338
Our results indicate that the Thr-197 phosphate of native, recombinant C subunit can be removed by a protein phosphatase activity found in extracts of S49 mouse lymphoma cells
This dephosphorylation could be monitored by either a mobility shift in SDS-PAGE or a reduction in protein kinase activity, and it resulted in loss of labeled phosphothreonine from C subunit autophosphorylated in the presence of [␥-32P]ATP
Summary
Catalytic (C) subunit of cAMP-dependent protein kinase when isolated from animal tissues is phosphorylated at two sites, Thr-197 and Ser-338. Comparisons of the structures of inactive forms of the CDC2 and MAP kinases with that of the active C subunit suggest that phosphorylation of sites in this “activation loop” region is responsible for a structural rearrangement of the catalytic cleft that promotes substrate binding and catalysis [4, 7]. While undertaking experiments to optimize conditions for immunoprecipitation of C subunit from extracts of S49 mouse lymphoma cells, we noted that incubation of radiolabeled, recombinant C subunit with cell extract resulted in apparent dephosphorylation manifested by an increase in SDS-PAGE mobility. This reaction was most dramatic in buffers containing SDS or urea, there was reactivity in the absence of these agents. This report describes experiments that identify the cellular activity as a protein phosphatase with properties similar to those of a multimeric form of protein phosphatase-2A (PP-2A) and characterize its reaction on C subunit
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