Effects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates

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Abstract
Reticulocyte lysates contain 2 major classes of protein phosphatase activities, designated type 1 and type 2. These designations are based on criteria derived from the analyses of protein phosphatase species in other tissues. The criteria include chromatographic elution profiles on DEAE-cellulose; specificity of lysate phosphatases toward [32P]phosphorylase a and [32P]phosphorylase kinase; sensitivity of lysate phosphatases to Mg2+ ATP; and sensitivity to the heat-stable protein phosphatase inhibitor-2. The lysate phosphatase species are similar to those described in rabbit skeletal muscle and rabbit liver. Reticulocyte protein phosphatase type 1, but not type 2, is inhibited by heat-stable protein phosphatase inhibitor-1 and -2 which have been characterized from rabbit skeletal muscle. The function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis were studied by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the .alpha.-subunit (38,000 daltons) of eIF-2 (eIF-2.alpha.) and of 2 heat-stable polypeptides of 29,000 and 44,000 daltons. The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significiance of this modification of inhibitor-2 is not clear. Inhibitor-2 has no effect on the activation in vitro of isolated heme-regulated or double-stranded RNA-dependent eIF-2.alpha. kinases. The inhibition of protein synthesis in hemin-supplemented lysates by added inhibitor-2 is due at least in part to the inhibition of a type I eIF-2.alpha. phosphatase activity, which permits a basal eIF-2.alpha. kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2.alpha. and an inhibition of protein synthesis.