Serine protein kinase activity associated with rotavirus phosphoprotein NSP5.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.     

Stromal serine protein kinase activity in spinach chloroplasts

At least twelve 32P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma-32P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma.     

Protein phosphorylation in Escherichia coli and purification of a protein kinase

More than 40 protein species including RNA polymerase were found to be phosphorylated in Escherichia coli on analyses of 32P-labeled cell lysates by single and two-dimensional gel electrophoresis and autoradiography. The protein species and the level of phosphorylation varied depending on the cell growth phase. With [gamma-32P]ATP as a substrate, cell lysates phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. Both serine and threonine were the major phosphate acceptors in whole cell lysates. Starting from a partially purified RNA polymerase preparation with the protein phosphorylation activity and using an E. coli protein with an apparent Mr = 90K (K represents X 1000) as the substrate, we purified a protein kinase with a native Mr approximately 120K to apparent homogeneity. The protein kinase is either a heterodimer of 61K and 66K polypeptides or a homodimer of one of these polypeptides. We also isolated a 100K protein with self-phosphorylation activity.     

A novel C-terminal proteolytic processing of cytosolic pyruvate kinase,its phosphorylation and degradation by the proteasome in developing soybean seeds

Cytosolic pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is an important glycolytic enzyme, but the post-translational regulation of this enzyme is poorly understood. Sequence analysis of the soybean seed enzyme suggested the potential for two phosphorylation sites: site-1 (FVRKGS220DLVN) and site-2 (VLTRGGS407TAKL). Sequence- and phosphorylation state-specific antipeptide antibodies established that cytosolic pyruvate kinase (PyrKinc) is phosphorylated at both sites in vivo. However, by SDS-PAGE, the phosphorylated polypeptides were found to be smaller (20-51 kDa) than the full length (55 kDa). Biochemical separations of seed proteins by size exclusion chromatography and sucrose-density gradient centrifugation revealed that the phosphorylated polypeptides were associated with 26S proteasomes. The 26S proteasome particle in developing seeds was determined to be of approximately 1900 kDa. In vitro, the 26S proteasome degraded associated PyrKinc polypeptides, and this was blocked by proteasome-specific inhibitors such as MG132 and NLVS. By immunoprecipitation, we found that some part of the phosphorylated PyrKinc was conjugated to ubiquitin and shifted to high molecular mass forms in vivo. Moreover, recombinant wild-type PyrKinc was ubiquitinated in vitro to a much greater extent than the S220A and S407A mutant proteins, suggesting a link between phosphorylation and ubiquitination. In addition, during seed development, a progressive accumulation of a C-terminally truncated polypeptide of approximately 51 kDa was observed that was in parallel with a loss of the full-length 55 kDa polypeptide. Interestingly, the C-terminal 51 kDa truncation showed not only pyruvate kinase activity but also activation by aspartate. Collectively, the results suggest that there are two pathways for PyrKinc modification at the post-translational level. One involves partial C-terminal truncation to generate a 51 kDa pyruvate kinase subunit which might have altered regulatory properties and the other involves phosphorylation and ubiquitin conjugation that targets the protein to the 26S proteasome for complete degradation.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.     

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.     

Protein kinases from Aspergillus nidulans that phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II.

Three serine kinases which phosphorylate the CTD of RNA polymerase II have been identified in Aspergillus nidulans. The kinases (KI, KII, KIII) were identified using a synthetic peptide containing four copies of the CTD consensus heptamer repeat, and differ in chromatographic behavior, and apparent molecular mass (KI approximately 60kDa; KII approximately 82kDa; KIII approximately 43 kDa). KIII utilized, in addition to peptide, histone H1 as substrate, whereas casein was not phosphorylated by any of the three kinases. The kinases appear to be unrelated to the p34cdc2 kinase, as judged by Western blot analysis and the position of serine phosphorylation of the synthetic CTD peptide. KI was highly purified and renaturation experiments have shown that it consists of a single polypeptide of 57 kDa. KI also phosphorylated RNA polymerase II associated in a preinitiation complex.     

An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver

1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [gamma-(32)P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [gamma-(32)P]ATP was markedly stimulated by Mg(2+), but not by cyclic AMP, cyclic GMP or Ca(2+). The phosphorylation of certain polypeptides was preferentially stimulated by Mg(2+). Addition of cyclic AMP resulted in a decrease in the amount of (32)P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [gamma-(32)P]GTP was found to be a relatively poor donor of (32)P as compared with [gamma-(32)P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [gamma-(32)P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of (32)P from [gamma-(32)P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of (32)P into this lipid was also markedly stimulated by Mg(2+). 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.     

The rate and extent of phosphorylation of the two light-harvesting chlorophyll a/b binding protein complex (LHC-II) polypeptides in isolated spinach thylakoids

The level of phosphorylation of the 24 kDa and the 25 kDa light-harvesting chlorophyll a/b binding protein complex (LHC) II polypeptides in isolated spinach thylakoids has been determined by quantitative SDS-polyacrylamide gel electrophoresis. The time-course of phosphorylation, after correction for the molar abundance of these two polypeptides, shows that (a) the rate of phosphorylation of the 24 kDa polypeptide is at least 3-fold faster compared with the 25 kDa polypeptide, (b) the final extent of phosphorylation for both the polypeptides is very similar, and (c) the final extent of phosphorylation is typically between 0.15 and 0.25 mol phosphate per mol polypeptide. The low extent of phosphorylation is not simply a consequence of inactivation of the kinase and / or LHC II substrate or ATP depletion. These observations suggest that there are at least three different sub-populations of LHC II in isolated thylakoids: (i) phosphorylated ‘mobile’, (ii) phosphorylated ‘PS II-coupled’ and (iii) non-phosphorylated. Furthermore, the reported differences in the specific activity of phosphorylation for the ‘mobile’ and the ‘PS II-coupled’ LHC II sub-populations (Kyle, D.J. et al. (1984) Biochim. Biophys. Acta 765, 89–96) are no longer observed following correction for the non-phosphorylated LHC-II population.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Phosphorylation of specific polypeptides in isolated murine splenocyte nuclei which is controlled by cyclic nucleotides

Murine splenocyte nuclei were phosphorylated with a less than 10(-5) M concentration of [gamma-32P]ATP at 0 degrees C and the phosphorylated nuclear proteins were analyzed by SDS-polyacrylamide gel slab electrophoresis and Sephadex gel filtration column chromatography. Two polypeptides of 10K and 11K daltons were predominantly phosphorylated. These polypeptides were likely linked by a disulfide bond to form a nonhistone protein of 21K daltons. Both phosphoserine and phosphothreonine were detected in the hydrolysate of the 10.5K dalton polypeptide, while phosphoserine was predominant in the 10K dalton polypeptide. Maximal activation of phosphorylation by cAMP of both polypeptides was shown at a concentration of 10(-6) M. On the contrary, cGMP activated phosphorylation of the 10K dalton polypeptide at 10(-8) M and at 10(-4) M. The phosphorylation of the 10.5K polypeptide was not activated by 10(-4) M cGMP and suppression of the phosphorylation was seen in both polypeptide chains by cAMP at higher concentrations.     

Evidence for an ectophosvitin kinase activity that phosphorylates a 123 kDa endogenous substrate on a human colonic adenocarcinoma cell (HT 29)

When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kinase activities. We have characterized one of them as an ectophosvitin kinase which phosphorylated specifically a 123 kDa polypeptide and whose expression or accessibility varied according to cell density.     

In vivo and in vitro acylation of polypeptides in Vibrio harveyi: identification of proteins involved in aldehyde production for bioluminescence.

Incubation of soluble extracts from Vibrio harveyi with [3H]tetradecanoic acid (+ ATP) resulted in the acylation of several polypeptides, including proteins with molecular masses near 20 kilodaltons (kDa), and at least five polypeptides in the 30- to 60-kDa range. However, in growing cells pulse-labeled in vivo with [3H]tetradecanoic acid, only three of these polypeptides, with apparent molecular masses of 54, 42, and 32 kDa, were specifically labeled. When extracts were acylated with [3H] tetradecanoyl coenzyme A, on the other hand, only the 32-kDa polypeptide was labeled. When luciferase-containing dark mutants of V. harveyi were investigated, acylated 32-kDa polypeptide was not detected in a fatty acid-stimulated mutant, whereas the 42-kDa polypeptide appeared to be lacking in a mutant defective in aldehyde synthesis. Acylation of both of these polypeptides also increased specifically during induction of bioluminescence in V. harveyi. These results suggest that the role of the 32-kDa polypeptide is to supply free fatty acids, whereas the 42-kDa protein may be responsible for activation of fatty acids for their subsequent reduction to form the aldehyde substrates of the bioluminescent reaction.     

Lipopolysaccharide-mediated induction of RNA polymerase I activity and amount in murine B lymphocytes

The effect of lipopolysaccharide on RNA polymerase I activity in primary cultures of murine B lymphocytes has been examined. In cells treated with mitogen for 48 h, the activity of RNA polymerase I was approximately 15 times greater than in control cells. In situ localization of RNA polymerase I using indirect immunofluorescence indicated that there was at least a 10-fold increase in the amount of this enzyme associated with nucleoli of 48 h mitogen-treated cells relative to control cells. Immunoblotting experiments demonstrated a similar increase in the concentration of the 190-kDa subunit bound to DNA; the concentrations of the other polymerase I-associated polypeptides did not correlate with rRNA synthesis. Assuming 1 mol of the 190-kDa polypeptide/mol of polymerase I, it was estimated that 2,300 and 30,000 molecules of enzyme were associated with rDNA in the unstimulated and stimulated B cell, respectively. Thus, an increased cellular concentration of the 190-kDa subunit of RNA polymerase I and its association with ribosomal DNA may be a crucial step in rRNA synthesis.