Vivian Ernst

REGULATION OF EUKARYOTIC PROTEIN CHAIN INITIATION BY PROTEIN KINASES AND HEME

Publisher Summary This chapter describes the regulation of eukaryotic protein chain initiation by protein kinases and heme. Reticulocyte lysates provide a model system for studies on the regulation of protein synthesis owing to several unique properties. They include an efficient rate of initiation, the extensive synthesis of one major protein species, and the complete dependence of protein synthesis on exogenous hemin. In the presence of optimal concentrations of added hemin, protein synthesis proceeds at linear rates for prolonged periods. During the period of linear synthesis, the polyribosomes are maintained, and initiation factors are utilized catalytically. In efficient lysates, the rate of globin synthesis is comparable to the in vivo rate. In the absence of added hemin, protein synthesis proceeds at the control rate for several minutes, and then declines abruptly yielding characteristic biphasic kinetics of inhibition. The studies have established that the inhibition is mediated through a translational inhibitor which is rapidly activated in the absence of heme.

EFFECTS OF THE CATALYTIC SUBUNIT OF PROTEIN KINASE II FROM RETICULOCYTES AND BOVINE HEART MUSCLE ON PROTEIN PHOSPHORYLATION AND PROTEIN SYNTHESIS IN RETICULOCYTE LYSATES

Publisher Summary This chapter describes the effects of the catalytic subunit of protein kinase II from reticulocytes and bovine heart muscle on protein phosphorylation and protein synthesis in reticulocytes lysates. The inhibition of protein synthesis initiation in heme-deficient lysates is caused by the activation of a heme-regulated cAMP-independent protein kinase (HRI), which phosphorylates the a-subunit of eIF-2 (eIF-2a). The findings suggest that the phosphorylation of HRI is related to its activation and is correlated with the phosphorylation of eIF-2a both in vitro and in situ. In an earlier study, it was observed that high levels of BHM protein kinase II (PK) plus cAMP (10−4M) partially inhibited protein synthesis in lysates. In an experiment described in the chapter, as cAMP alone has no effect on protein synthesis in lysates, the effects of highly purified catalytic subunits of PK II from reticulocytes (rC) and BHM (bC) were examined. Both rC and bC migrated as 43,000 dalton polypeptides in SDS-PAGE and were autophosphorylated when preincubated with [γ−32P]ATP. The addition of rC (or bC) to lysates at physiological concentrations does not affect protein synthesis, but at five- to tenfold higher levels, some inhibition (20–25%) was also observed.

THE IN SITU PHOSPHORYLATION OF THE α-SUBUNIT OF eIF-2 IN RETICULOCYTE LYSATES INHIBITED BY HEME DEFICIENCY, dsRNA, GSSG, OR THE HEME-REGULATED INHIBITOR

Publisher Summary This chapter analyzes in situ phosphorylation of the α-subunit of eIF-2 in reticulocyte lysates inhibited by heme deficiency, dsRNA, GSSG, or the heme-regulated inhibitor. The initiation of protein synthesis in reticulocyte lysates is inhibited in the absence of hemin, or in the presence of GSSG (50–500 μM) or dsRNA (1–50 ng/ml). All three conditions activate cAMP-independent protein kinases that phosphorylate the α-subunit (38,000 daltons) of eIF-2 (elF-2α). In this study, this phosphorylation is examined directly in lysates by labeling for brief periods with a pulse of high specific activity [γ-32P]ATP. The [32P]phosphoprotein profiles were analyzed by one-dimensional SDS-PAGE and autoradiography under conditions in which the eIF-2α polypeptide could be clearly distinguished. All modes of inhibition produced a rapid and significant increase in the phosphorylation of eIF-2α, compared to uninhibited control incubations which displayed little or no phosphorylation of eIF-2α. In heme-deficient lysates, phosphorylation of eIF-2α was detectable within 30 seconds after the start of incubation, and at all times subsequent both before and after the shut-off of protein synthesis. The restoration of synthesis by the delayed addition of hemin is accompanied by a decrease in the phosphorylation of eIF-2α.