The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle

Abstract

Inhibitor‐2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases‐1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases‐1 and 2A accounted for all the inhibitor‐2 phosphatases activity in the absence of Ca²⁺(resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase‐2B, a Ca²⁺‐calmodulin‐dependent enzyme, could account for up to 30% of the inhibitor‐2 phosphatase activity in contracting muscle. The K_m of protein phosphatase‐1 for inhibitor‐2(40 nM) was 100‐fold lower than the K_m for phosphorylase a (4.8 μM). This finding, coupled with the failure of inhibitor‐2 to inhibit its own dephosphorylation, suggests that inhibitor‐2 is dephosphorylated at one of the two sites on protein phosphatase‐1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor‐2 by protein phosphatase‐1 was also uñaffected by inhibitor‐1, suggesting that the phosphorylation state of inhibitor‐2 is unlikely to be controlled by cyclic AMP in vivo.