Open AccessEffects of skeletal muscle protein phosphatase inhibitor-2 on protein synthesis and protein phosphorylation in rabbit reticulocyte lysates
Author(s) -
Vivian Ernst,
Daniel H. Levin,
J G Foulkes,
Irving M. London
Publication year - 1982
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.79.23.7092
Subject(s) - reticulocyte , phosphatase , biochemistry , biology , lysis , protein phosphatase 1 , microbiology and biotechnology , phosphorylation , protein biosynthesis , protein kinase a , protein phosphorylation , messenger rna , gene
Reticulocyte lysates contain two 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 (i ) chromatographic elution profiles on DEAE-cellulose; (ii ) specificity of lysate phosphatases toward [32 P]phosphorylasea and [32 P]phosphorylase kinase; (iii ) sensitivity of lysate phosphatases to Mg2+ ATP; and (iv ) 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. We have initiated a study on the function and specificity of lysate protein phosphatase activities involved in the regulation of protein synthesis by examining the effects of protein phosphatase inhibitor-2 on reticulocyte protein synthesis and protein phosphorylation. Our findings are as follows. (a ) Protein phosphatase inhibitor-2 inhibits protein chain initiation in hemin-supplemented lysates. (b ) Inhibition is characterized by biphasic kinetics and is reversed by the delayed addition of purified reticulocyte eukaryotic initiation factor 2 (eIF-2). (c ) Inhibition of protein synthesis by inhibitor-2 is accompanied by the phosphorylation of the α-subunit (38,000 daltons) of eIF-2 (eIF-2α) and of two heat-stable polypeptides of 29,000 and 44,000 daltons. (d ) The 29,000-dalton component is phosphorylated in lysates under conditions of protein synthesis and appears to be inhibitor-2, but the physiological significance of this modification of inhibitor-2 is not clear. (e ) Inhibitor-2 has no effect on the activationin vitro of isolated heme-regulated or double-stranded RNA-dependent eIF-2α kinases. We propose that 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 1 eIF-2α phosphatase activity, which permits a basal eIF-2α kinase activity to be expressed leading to the accumulation of phosphorylated eIF-2α and an inhibition of protein synthesis.