Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus.

Abstract

We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16 It is well recognized that regulation of enzyme activity may be achieved by several distinct mechanisms. In recent years evidence has accumulated implicating an enzymatic interconversion of active and inactive forms of enzymes as a major regulatory control mechanism. The activity of an increasing number of enzymes may be regulated and modulated through phosphorylation or dephosphorylation involving the coordinated action of protein phosphokinases and phosphoprotein phosphatases on their enzyme substrates (1-7). The opportunities that such systems provide for coordinated control of enzymic activity are amply illustrated by the now well-established phosphorylative and functional modifications of glycogen synthetase and glycogen phosphorylase (2, 3).There is evidence that infection and transformation of susceptible cells by oncogenic RNA viruses, such as Rous sarcoma virus (RSV), is critically dependent upon the activity of the reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) (8,9). In view of this finding, it is of importance to determine whether or not the enzymatic activity of the reverse transcriptase may be altered and modified and whether the modification occurs as a consequence of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) action. In the present communication we wish to report that protein phosphokinase isolated from RSV-transformed chick embryo fibroblasts can enhance reverse transcriptase activity of Rous sarcoma virus and that the increase in activity can be reversed through the action of phosphatase. Cells and Viruses. Schmidt-Ruppin RSV, subgroup D, (SR-RSV-D) was grown in type c/o chick embryo fibroblasts and purified as described (11). Two weeks after infection by SR-RSV-D, the transformed cells were removed with scraping, washed with a solution of 0.14 M NaCl/0.1 M sodium phosphate, pH 7.2, and pelleted by centrifugation at 1000 X g. The recovered cells were used for the isolation of protein kinase.Enzyme Preparations. Protein kinase from the cytosol (105,000 X g supernatant fraction) of RSV-transformed cells was isolated and purified as described (6). After DEAE-cellulose chromatography, the pooled fractions containing protein kinase activity were additionally purified by Sephadex G-200 gel filtration (elution buffer: 0.05 M Tris buffer, pH 7.2, containing 2 mM MgCl2, and 1 mM 2-mercaptoethanol) followed by preparative isoelectric focusing (12). The protein kinase preparation used in these studies had a specific activity of 11.6 pmol of 32P incorporated/mg of protein per min using protamine sulfate as substrate. Addition of cyclic AMP (1 1,M) to this kinase preparation resulted in a 1.54-fold stimulation of the kinase activity.Reverse transcriptase from RS-RSV-D was purified by the method of Grandgenett et al. (13). The major peak of r...

Influence of phosphate on activity and stability of reverse transcriptase from avian myeloblastosis virus

“…In a similar experiment ( Fig. 2A) (5). These results show that the influence of phosphate observed in the maintenance of RDDP activity (Fig.…”

“…One of the most widely studied modifications is protein phosphorylation-dephosphorylation (1). The enzymatic machinery that can carry out this protein modification in AMV and other oncornaviruses has been identified (2)(3)(4) and recent observations in vitro (5) indicate that RDDP from Rous sarcoma virus (RSV) may be one of these modified proteins. The possibility exists that phosphorylation and dephosphorylation occurs with RDDP of other and perhaps all oncornaviruses and thus may constitute a regulatory mechanism of considerable biological significance.…”

Protein kinase from avian myeloblastosis virus

“…In consequence, limitations on the ultimate usefulness of the enzyme are dependent in large part on the provision and maintenance of the full integrity of its capabilities. In this respect, it has been reported that a protein kinase from Rous sarcoma virus-transformed chicken embryo fibroblasts in tests in vitro effected an increase in Rous sarcoma virus transcriptase activity (11). More recently, it was shown that the DNA polymerase of the B77 strain of Rous sarcoma virus was phosphorylated, and that the phosphorus was associated with the f8 component of the transcriptase (6).…”

“…Of the nonvirus proteins tested, only the two acidic proteins, a-casein and phosvitin, accepted phosphate. In view of recent reports (11,25,26) describing phosphorylation and associated increase in the activity of RDDP purified from AMV or Rous sarcoma virus, we assayed a series of 12 different preparations each of 90% or more pure RDDP (Houts et al, in preparation) for the ability to accept phosphate. With all except one preparation, RDDP was present in the reaction mixtures in concentrations of 27 to 134 ,ug/ml; however, one of the preparations (G-677) was further concentrated and used in the reaction at 810 ,ug/ml.…”

“…Protein kinase occurs in a variety of enveloped animal viruses (4, 5, 7, 9-11, 13-21, 23-25, 27) and has been isolated in purified preparations from vaccinia (9,10) and frog virus (FV3) (19), of which the enzyme is a virion-specific component (20). The presence of protein kinase has been noted, also, in avian myeloblastosis virus (AMV) (18), and a recent publication (11) reported studies on phosphorylation of preparations of reverse transcriptase (RDDP) from Rous sarcoma virus by protein kinase extracted from Rous sarcoma virus-transformed chicken embryo fibroblasts. These results, together with possible applicability of protein kinase to developmental work in this laboratory, prompted continued exploration of procedures for isolation of the enzyme from AMV.…”