AMP-independent Protein Kinase Research Articles

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2αkinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3′:5′-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2α). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2α. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2α is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2α kinase activities which are inhibitory of protein synthesis.

Evidence for two protein kinases associated with the surface membranes of L-929 cells

L-929 cell surface membranes have been assayed in vitro and found to contain significant protein kinase activity. A steady-state kinetic analysis indicated that at least two distinct protein kinases were present. Plots of reaction velocity ( v) against substrate (ATP) concentration were distinctly biphasic, as were Lineweaver-Burk plots of 1 v versus 1 ATP . Michaelis constants of the two enzymes were calculated to be 22 and 173 μ m, respectively. Sodium dodecyl sulfate polyacrylamide gel analysis of the phosphorylated membrane proteins provided additional support for the existence of more than one protein kinase. Different endogenous proteins were phosphorylated at 1 μ m ATP compared to 1 μ m ATP. Further studies of the low K m (22 μ m) enzyme suggested that it is a typical cyclic 3′,5′-AMP-independent protein kinase. Its activity was dependent on the presence of Mg 2+, but it was not affected by cyclic 3′,5′-AMP, cyclic 3′,5′-GMP, or the heat-stable inhibitor of cyclic 3′,5′-AMP-dependent protein kinases. ATP and GTP, but not other nucleoside triphosphates, could serve as phosphoryl donor and maximum kinase activity was expressed at pH 7.0. Phosvitin and casein were superior to histones as exogenous substrates for the low K m enzyme.

Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates

Double-stranded RNA (dsRNA) activates a cyclic 3′: 5′-AMP independent protein kinase (dsI) in reticulocyte lysates which inhibits protein synthesis by phosphorylating the 38, 000 dalton (38K) subunit of the initiation factor eIF-2 (eIF-2α). A latent precursor form of dsI (latent dsI) has been partially purified (1000–2000 fold) from lysates. Activation of dsI at all stages in the purification of latent dsI requires ATP and low levels of dsRNA (1–20 ng/ml), and is accompanied by the phosphorylation of a broad 67,000 dalton (67K) band. However, as purification proceeds the 67K band is resolved into two phosphorylated polypeptides of 68,500 and 67,000 daltons ( 68.5K 67K ). Although latent dsI and activated dsI have distinctly different chromatographic properties, both forms have similar molecular weights (∼120,000) and similar sedimentation coefficients (∼3.8S) in glycerol gradients. The data support the view that one or both components of the 68.5K 67K doublet are associated with the dsRNA-dependent protein kinase activity.

Partial purification and properties of a cyclic 3′,5′-ampindependent protein kinase from rat liver

1. 1. A cyclic 3′,5′-AMP-independent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from rat liver cytosol was partially purified and characterized. Purification by (NH 4) 2SO 4 precipitation, DEAE-cellulose, Bio Gel A-0.5 m and cellulose phosphate chromatography increased the specific activity about 700-fold. 2. 2. An endogenous protein substrate was closely associated with the protein kinase and was not separable from this enzyme up to the cellulose phosphate stage. After phosphorylation, chromatography with Bio Gel A-0.5 m partially separated this endogenous phosphoprotein from the enzyme activity; this dissociation had no apparent effect on kinase activity with casein or phosvitin as substrates, or on the apparent molecular weight of the enzyme (approx. 158 000). 3. 3. This protein kinase with casein, phosvitin, or the endogenous substrate was totally insensitive to the thiol reagents, p- hydroxymercuribenzoate , 5,5′ dithiobis(2-nitrobenzoic acid), iodoacetamide, and N- ethylmaleimide . The enzyme was also unaffected by cyclic 3′,5′-AMP, heat-stable protein kinase inhibitor, and the regulatory subunit of a cyclic 3′,5′-AMP-dependent protein kinase.