DEPHOSPHORYLATION OF CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE AT THR-197 BY A CELLULAR PROTEIN PHOSPHATASE AND BY PURIFIED PROTEIN PHOSPHATASE-2A
S. Liauw et Ra. Steinberg, DEPHOSPHORYLATION OF CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE AT THR-197 BY A CELLULAR PROTEIN PHOSPHATASE AND BY PURIFIED PROTEIN PHOSPHATASE-2A, The Journal of biological chemistry, 271(1), 1996, pp. 258-263
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.