HPr, a phosphate carrier protein of the streptococcal phosphotransferase system, is phosphorylated at the N-1 position of a single histidyl residue in a reaction requiring phosphoenolpyruvate (P-ePrv), Mg2+, and enzyme I (P-ePrv-HPr phosphotransferase, EC2.7.3.9). We demonstrate that in addition to this reaction, a seryl residue within HPr can be phosphorylated in an ATP-dependent process. This reaction is catalyzed by a protein kinase with an approximate Mr of 20,000. In whole cells the kinase activity is stimulated by glucose, whereas in crude extracts the activity is stimulated by glycolytic intermediates such as glucose 6-phosphate, fructose 1,6-diphosphate, and 2-phosphoglycerate. P-(Ser)-HPr cannot transfer its phosphate group via enzyme II to a sugar as does the P-(His)-HPr. Instead, a phosphatase (Mr = 70,000) was found to hydrolyze the phosphate group of P-(Ser)-HPr. The phosphatase reaction is strongly inhibited by the addition of P-ePrv and enzyme I. Protein kinase-catalyzed phosphorylation of the enzyme constituents of the phosphotransferase system in Escherichia coli has also been demonstrated. These observations lead us to suggest that phosphorylation of a seryl residue in HPr is involved in the regulation of sugar transport in the bacteria cell.

https://www.pnas.org/content/pnas/80/22/6790.full.pdf

ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes

We report on phosphorylation, the stability of the bound phosphate, and the properties of several phosphorylated infected-cell polypeptides (ICPs) synthesized in cells infected with herpes simplex virus 1 and 2. Our results and conclusions are as follows. (i) Phosphorylation of ICPs occurs by at least two different pathways. Thus, the 4a and 4c electrophoretic forms of ICP 4 were labeled with 32P during a pulse concurrently with their synthesis, whereas ICP 22 and ICP 27 were labeled with 32P only during a subsequent chase in the presence of unlabeled phosphate. (ii) Pulse chase studies with [35S]methionine and 32P indicate that whereas most polypeptides are stable, the bound phosphate with few exceptions cycles on and off. Of special interest is the observation that the phosphate bound to ICP 4a and 4c cycles on and off, whereas that bound to ICP 4b is stably associated. Similar cycling was observed for ICP 6, 11, 22, and 27. The observation that 4a and 4c can be phosphorylated as late as 24 h after infection, i.e., long after their synthesis ceases, suggests that all three forms may have defined functions that persist throughout the reproductive cycle. (iii) All three forms of ICP 4 can be the translational products of only one of two copies of the ICP 4 gene in the viral genome. (iv) Analyses of the distribution of the viral proteins within the cell indicate that phosphorylation is not a major determinant in the compartmentalization of most viral phosphoproteins. (v) Comparisons of the binding to DNA-cellulose of artificial mixtures of 32P- and [35S]methionine-labeled proteins from infected cells indicate that phosphorylation in some instances enhances (e.g., ICP 29) and in other instances decreases (e.g., ICP 6) binding affinity for DNA. In light of previous reports that some of the proteins identified as phosphoproteins have regulatory functions, the data suggest that phosphorylation may modify the activity of regulatory proteins in herpes simplex virus-infected cells.

Herpes simplex virus phosphoproteins. I. Phosphate cycles on and off some viral polypeptides and can alter their affinity for DNA.

Molecular Evolution of Multisubunit RNA Polymerases: Sequence Analysis

Phosphorylation of eukaryotic DNA-dependent RNA polymerase. Identification of calf thymus RNA polymerase subunits phosphorylated by two purified protein kinases, correlation with in vivo sites of phosphorylation in HeLa cell RNA polymerase II.

The first clearly established example of Ser/Thr/Tyr phosphorylation of a bacterial protein was isocitrate dehydrogenase. In 1979, 25 years after the discovery of protein phosphorylation in eukaryotes, this enzyme was reported to become phosphorylated on a serine residue. In subsequent years, numerous other bacterial proteins phosphorylated on Ser, Thr or Tyr were discovered and the corresponding protein kinases and P-protein phosphatases were identified. These protein modifications regulate all kinds of physiological processes. Ser/Thr/Tyr phosphorylation in bacteria therefore seems to play a similar important role as in eukaryotes. Surprisingly, many bacterial protein kinases do not exhibit any similarity to eukaryotic protein kinases, but rather resemble nucleotide-binding proteins or kinases phosphorylating diverse low-molecular-weight substrates.

Ser/Thr/Tyr Protein Phosphorylation in Bacteria – For Long Time Neglected, Now Well Established

After infection with bacteriophage T7 the beta' and to a lesser extent the beta subunits of E. coli DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) are phosphorylated by a phage-gene-encoded protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The phosphorylation occurs on threonine residues and appears site-specific. It is probably the molecular basis of the early transcriptional control.

http://www.pnas.org/content/72/7/2506.full.pdf

Werner Blum

Papers

In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.