The protein phosphatases involved in cellular regulation

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Abstract
Protein phosphatases‐2Ao, 2A1and 2A2have been purified to homogeneity from rabbit skeletal muscle. Approximately 1 mg of phosphatase‐2A0and 2A1, and 0.5 mg of phosphatase‐2A2, was isolated from 4000 g muscle within 10 days. Protein phosphatases‐2Aoand 2A1each comprised three subunits, termed A, B' and C (2Ao) or A, B and C (2A1), while phosphatase‐2A2contained only two subunits, A and C. The A and C components of phosphatases‐2Ao, 2A1and 2A2had indistinguishable mobilities on sodium dodecyl sulphate/ polyacrylamide gels and identical peptide maps. By these criteria, the C component was also identical to the catalytic subunit of phosphatase‐2A purified from ethanol‐treated muscle extracts. The electrophoretic mobilities of the B and B′ subunits were slightly different, and their peptide maps were distinct.The molecular masses of the native enzymes determined by sedimentation equilibrium centrifugation were 181 ± 6 kDa (2Ao), 202 ± 6 kDa (2A1) and 107 ± 5 kDa (2A2), while those of the subunits estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis were 60 kDa (A), 55 kDa (B), 54 kDa (B′) and 36 kDa (C). These values, in conjunction with molar ratios estimated by densitometric analyses of the gels, suggest that the subunit structures of the enzymes are AB'C2(2Ao), ABC2(2A1) and AC (2A2). Protein phosphatase‐2A2appears to be derived from 2Aoand/or 2A1during purification through degradation or dissociation of the B′ and/or B subunits.Protein phosphatases‐2Ao, 2A1and 2A2were the only phosphorylase phosphatases in rabbit skeletal muscle that were activated by the basic proteins, protamine (A0.5= 0.25 μM), histone H 1 (A0.5= 0.3 μM) and polylysine (A0.5= 0.04 μM). Activation by protamine varied over 5–20‐fold for phosphatase‐2Aoand 5–7‐fold for phosphatases‐2A1and 2A2. The dephosphorylation of glycogen synthase was activated by basic proteins in a similar manner to the phosphorylase phosphatase activity. The isolated C subunit was also stimulated by histone H1 and protamine, but 5–10‐fold higher concentrations were required, and with phosphorylase as substrate, maximum activation was only about 2‐fold. Activation by basic proteins appears to involve their interaction with the A and/or C subunits, but not with the B or B′ subunits, or substrates phosphorylase and glycogen synthase.

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