The GTP-binding protein YqeH participates in biogenesis of the 30S ribosome subunit in Bacillus subtilis

Bacterial genome sequencing has revealed a novel family of P-loop GTPases that are often essential for growth. Accumulating evidence suggests that these proteins are involved in biogenesis of the 30S or 50S ribosomal subunits. YqeH is a member of this Obg/Era GTPase family, with its function remains to be uncovered. Here, we present results showing that YqeH is involved in the 30S subunit biogenesis in Bacillus subtilis. We observed a reduction in the 70S ribosome and accumulation of the free 50S subunit in YqeH-depleted cells. Interestingly, no free 30S subunit accumulation was evident. Consistent with the theory that YqeH is involved in 30S subunit biogenesis, a precursor of 16S rRNA and its degradation products were detected. Additionally, the reduction of free 30S subunit was not observed in Era-depleted cells. YqeH overexpression did not compensate for growth defects in mutants devoid of Era and vice versa. Moreover, in vitro GTPase analyses showed that YqeH possessed high intrinsic GTPase activity. In contrast, Era showed slow GTPase activity, which was enhanced by the 30S ribosomal subunit. Our findings strongly suggest that YqeH and Era function at distinct checkpoints during 30S subunit assembly. B. subtilis yqeH is classified as an essential gene due to the inability of the IPTG-dependent P(spac)-yqeH mutant to grow on LB or PAB agar plates in the absence of IPTG. However, in our experiments, the P(spac)-yqeH mutant grew in PAB liquid medium without IPTG supplementation, albeit at an impaired rate. This finding raises the interesting possibility that YqeH participates in assembly of the 30S ribosomal subunit as well as other cellular functions essential for growth on solid media.     

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.     

Isolation and characterization of a dominant negative mutant of Bacillus subtilis GTP-binding protein, YlqF, essential for biogenesis and maintenance of the 50 S ribosomal subunit

The circularly permuted GTPase YlqF is essential for cell viability and is broadly conserved from Gram-positive bacteria to eukaryotes. We previously reported that YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis. Here, we demonstrate that an N-terminal deletion mutant of YlqF (YlqFDeltaN10) inhibits cell growth even in the presence of wild-type YlqF. In contrast to the wild-type protein, the GTPase activity of this mutant was not stimulated by the 50 S subunit and did not dissociate from the premature 50 S subunit. Thus, YlqFDeltaN10 acts as a competitive inhibitor of wild-type YlqF. Premature 50 S subunit lacking ribosomal protein L27 and with a reduced amount of L16 accumulated in YlqFDeltaN10-overexpressing cells and in YlqF-depleted cells, suggesting that YlqFDeltaN10 binds to the premature 50 S subunit. Moreover, premature 50 S subunit from both YlqFDeltaN10-overexpressing and YlqF-depleted cells more strongly enhanced the GTPase activity of YlqF than the mature 50 S subunit of the 70 S ribosome. Collectively, our results indicate that YlqF is targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA.     

The GTPase function of YvcJ and its subcellular relocalization are dependent on growth conditions in Bacillus subtilis

We have recently shown that the Bacillus subtilis GTPase YvcJ is involved in the phosphorylation of an unidentified cellular component and that the deletion of yvcJ induced a decrease in competence efficiency. In this paper, we report that growth conditions influence both the YvcJ-dependent phosphorylation event and the localization of this protein. More precisely, we have observed that YvcJ can be localized in the cell either as a helical-like pattern or as foci close to the poles and the septa depending on growth phase and on growth medium. In addition, we show that the mutation of the catalytic lysine residue (K22) located in the Walker A motif of YvcJ, and necessary for its GTPase activity, induces a decrease in competence efficiency similar to that observed for the yvcJ null mutant. This mutation also inhibits the YvcJ-dependent phosphorylation event. Furthermore, a phylogenetic analysis of the YvcJ homologues shows that this protein is ancient in Bacteria (being possibly present in their last common ancestor) and has been conserved in a number of major bacterial phyla, suggesting that this protein has an important function in this domain of life. To sum up, even if the precise cellular role of this ancient protein remains unknown, our data show that the GTPase activity of B. subtilis YvcJ and its function in the phosphorylation of a cellular component are influenced by the growth conditions, and are important for the effect of YvcJ on competence efficiency.     

Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis

Although many membrane Ser/Thr‐kinases with PASTA motifs have been shown to control bacterial cell division and morphogenesis, inactivation of the Ser/Thr‐kinase PrkC does not impact Bacillus subtilis cell division. In this study, we show that PrkC localizes at the division septum. In addition, three proteins involved in cell division/elongation, GpsB, DivIVA and EzrA are required for stimulating PrkC activity in vivo. We show that GpsB interacts with the catalytic subunit of PrkC that, in turn, phosphorylates GpsB. These observations are not made with DivIVA and EzrA. Consistent with the phosphorylated residue previously detected for GpsB in a high‐throughput phosphoproteomic analysis of B. subtilis, we show that threonine 75 is the single PrkC‐mediated phosphorylation site in GpsB. Importantly, the substitution of this threonine by a phospho‐mimetic residue induces a loss of PrkC kinase activity in vivo and a reduced growth under high salt conditions as observed for gpsB and prkC null mutants. Conversely, substitution of threonine 75 by a phospho‐ablative residue does not induce such growth and PrkC kinase activity defects. Altogether, these data show that proteins of the divisome control PrkC activity and thereby phosphorylation of PrkC substrates through a negative feedback loop in B. subtilis.     

The GTP-binding protein YlqF participates in the late step of 50 S ribosomal subunit assembly in Bacillus subtilis

Bacillus subtilis YlqF belongs to the Era/Obg subfamily of small GTP-binding proteins and is essential for bacterial growth. Here we report that YlqF participates in the late step of 50 S ribosomal subunit assembly. YlqF was co-fractionated with the 50 S subunit, depending on the presence of noncleavable GTP analog. Moreover, the GTPase activity of YlqF was stimulated specifically by the 50 S subunit in vitro. Dimethyl sulfate footprinting analysis disclosed that YlqF binds to a unique position in 23 S rRNA. Yeast two-hybrid data revealed interactions between YlqF and the B. subtilis L25 protein (Ctc). The interaction was confirmed by the pull-down assay of the purified proteins. Specifically, YlqF is positioned around the A-site and P-site on the 50 S subunit. Proteome analysis of the abnormal 50 S subunits that accumulated in YlqF-depleted cells showed that L16 and L27 proteins, located near the YlqF-binding domain, are missing. Our results collectively indicate that YlqF will organize the late step of 50 S ribosomal subunit assembly.     

PrkC-mediated Phosphorylation of Overexpressed YvcK Protein Regulates PBP1 Protein Localization in Bacillus subtilis mreB Mutant Cells

The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state.     

Modification of yeast ribosomal proteins. Phosphorylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins labelled in vivo with 32PO43- revealed that the proteins S2 and S10 of the 40S ribosomal subunit, and the proteins L9, L30, L44 and L45 of the 60S ribosomal subunit, are phosphorylated in vivo. Most of the phosphate groups appeared to be linked to serine residues. Teh number of phosphate groups per molecule of phosphorylated protein species ranged from 0.01 to 0.79. Since most of the phosphorylated ribosomal proteins appear to associate with the pre-ribosomal particles at a very late stage of ribosome assembly, phosphorylation is more likely to play a role in the functioning of the ribosome than in its assembly.     

The activity of protein kinases from hamster fibroblasts towards a synthetic peptide based on a carboxy-terminal portion of ribosomal protein S6

A synthetic decapeptide, S6(231-240), based on a region near the C-terminus of eukaryotic ribosomal protein S6, was used as a substrate for protein kinases (EC 2.7.1.37) from hamster fibroblasts stimulated with fresh medium. Consistent with the results of others using shorter peptides from this region, it was found that the cyclic AMP-dependent protein kinase preferentially phosphorylated the residue corresponding to Ser-235, whereas protein kinase C preferentially phosphorylated the residue corresponding to Ser-236 in this peptide. The peptide did not serve as a substrate for the growth-associated protein kinase from hamster fibroblasts that phosphorylated ribosomal protein S6 in 40S ribosomal subunits, but did serve as a substrate for a previously undetected protein kinase activity that was resolved from the latter by DEAE-cellulose chromatography. This S6(231-240) protein kinase activity did not phosphorylate ribosomal protein S6 in 40S ribosomal subunits, but is possibly a proteolytic fragment of the 40S ribosomal subunit S6 kinase as the latter activity acquired the ability to phosphorylate the decapeptide after partial tryptic proteolysis. The S6(231-240) protein kinase activity preferentially phosphorylated the residue corresponding to Ser-236 with an apparent Km of 15 microM. These results suggest that specific interactions with the ribosome may be required to activate the growth-associated ribosomal protein S6 kinase.     

Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro

Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h(-1)) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41: 11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.     

Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC,a Ser/Thr kinase from Bacillus subtilis

We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant proteins were tested for in vitro kinase activity. Single and multiple replacement of four threonine residues, clustered between residues 162 and 167 in a putative activation loop, substantially reduced kinase activity and the effect was clearly additive. Replacement of the other three threonine residues, clustered between residues 290 and 320, had relatively little effect on activity. In contrast, substitution of Ser214, which is conserved in closely related receptor kinase-like bacterial proteins, independently affected activity and may represent a novel regulatory mechanism. When projected onto a 3D structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located in the juxtamembrane region. These results indicate that regulation of PrkC kinase activity (and presumably autophosphorylation) includes a conserved activation loop mechanism. The juxtamembrane phospho-threonine residues may be essential, for example for the recruitment of other proteins necessary for a PrkC signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family.     

Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3

Mammalian mitochondrial ribosomes synthesize 13 proteins that are essential for oxidative phosphorylation. In addition to their role in protein synthesis, some of the mitochondrial ribosomal proteins have acquired functions in other cellular processes such as apoptosis. Death-associated protein 3 (DAP3), also referred to as mitochondrial ribosomal protein S29 (MRP-S29), is a GTP-binding pro-apoptotic protein located in the small subunit of the ribosome. Previous studies have shown that phosphorylation is one of the most likely regulatory mechanisms for DAP3 function in apoptosis and may be in protein synthesis; however, no phosphorylation sites were identified. In this study, we have investigated the phosphorylation status of ribosomal DAP3 and mapped the phosphorylation sites by tandem mass spectrometry. Mitochondrial ribosomal DAP3 is phosphorylated at Ser215 or Thr216, Ser220, Ser251 or Ser252, and Ser280. In addition, phosphorylation of recombinant DAP3 by Protein kinase A and Protein kinase Cdelta at residues that are endogenously phosphorylated in ribosomal DAP3 suggests both of these kinases as potential candidates responsible for the in vivo phosphorylation of DAP3 in mammalian mitochondria. Interestingly, the majority of the phosphorylation sites detected in our study are clustered around the highly conserved GTP-binding motifs, speculating on the significance of these residues on protein conformation and activity. Site-directed mutagenesis studies on selected phosphorylation sites were performed to determine the effect of phosphorylation on cell proliferation and PARP cleavage as indication of caspase activation. Overall, our findings suggest DAP3, a mitochondrial ribosomal small subunit protein, is a novel phosphorylated target.     

The GTPase, CpgA(YloQ), a putative translation factor, is implicated in morphogenesis in Bacillus subtilis

YloQ, from Bacillus subtilis, was identified previously as an essential nucleotide-binding protein of unknown function. YloQ was successfully over-expressed in Escherichia coli in soluble form. The purified protein displayed a low GTPase activity similar to that of other small bacterial GTPases such as Bex/Era. Based on the demonstrated GTPase activity and the unusual order of the yloQ G motifs, we now designate this protein as CpgA (circularly permuted GTPase). An unexpected property of this low abundance GTPase was the demonstration, using gel filtration and ultracentrifugation analysis, that the protein formed stable dimers, dependent upon the concentration of YloQ(CpgA), but independent of GTP. In order to investigate function, cpgA was placed under the control of the pspac promotor in the B. subtilis chromosome. When grown in E or Spizizen medium in the absence of IPTG, the rate of growth was significantly reduced. A large proportion of the cells exhibited a markedly perturbed morphology, with the formation of swollen, bent or ‘curly’ shapes. To confirm that this was specifically due to depleted CpgA a plasmid-borne cpgA under pxyl control was introduced. This restored normal cell shape and growth rate, even in the absence of IPTG, provided xylose was present. The crystal structure of CpgA(YloQ) suggests a role as a translation initiation factor and we discuss the possibility that CpgA is involved in the translation of a subset of proteins, including some required for shape maintenance. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus

Ribosomal protein phosphorylation was investigated in Ehrlich ascites tumor cells infected with vaccinia virus (Copenhagen strain). After 90 min of simultaneous infection and 32P-labelling, ribosomal proteins Sa, S2 and S13 appear specifically phosphorylated as well as Sb/La, P1 and S6, which are also phosphorylated in control cells. Sa is an acidic protein, whose phosphorylation has not been observed previously. A kinetic study showed that S2 is phosphorylated very rapidly within 10 min after the beginning of infection and it is complete 1 h later. The phosphorylation of S13 begins after a lag time of about 1 h and is completed after about 2.5 h of infection. Moreover only one phosphate is incorporated into S13 on a serine residue while up to four phosphates are incorporated into S2, the first on a serine and the three following on threonine residues. In vivo experiments, carried out in the presence of cycloheximide and cordycepin, suggest a viral origin for the kinase involved in the phosphorylation of S2 and S13. Moreover, in vitro experiments demonstrated that the kinase associated with the viral cores is capable of phosphorylating S2 on a serine residue only. In our cell/virus system, no significant difference in S6 phosphorylation was detected, when compared to uninfected cells. It is concluded that the specific and efficient phosphorylation of three ribosomal proteins from the 40S ribosomal subunit correlate well with possible translational mechanisms ensuring the efficient expression of early and late genes of vaccinia virus. In the light of these and previous results [Person, A. and Beaud, G. (1986) J. Biol. Chem. 261, 8283-8289], a mechanism is proposed for the shut-off of host protein synthesis and the selective translation of mRNAs of viral origin.     

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Purification of a hepatic S6 kinase from cycloheximide-treated Rats

Cycloheximide injection of rats results in the activation of a protein kinase that phosphorylates 40 S ribosomal protein S6. This Ca2+/cyclic nucleotide-independent kinase exhibits chromatographic properties that are indistinguishable from the S6 kinase in H4 hepatoma cells whose activity is stimulated by insulin and growth factors and the S6 kinase that is activated during liver regeneration. The enzyme has been purified 50,000-fold to near homogeneity: a critical step in purification employs a peptide affinity column using a synthetic peptide corresponding to the carboxyl-terminal 32-amino acid residues of mouse liver S6, which encompasses all S6 phosphorylation sites. The purified enzyme is a 70,000-dalton polypeptide that is reactive with azido-ATP. In addition to 40 S ribosomal S6 and the synthetic peptide, the S6 kinase catalyzes rapid phosphorylation of a number of other protein substrates including histone H2b, glycogen synthase, and ATP citrate lyase; this last protein is phosphorylated by S6 kinase in vitro on the same serine residue that is phosphorylated in response to insulin and epidermal growth factor in intact hepatocytes. Moreover, the S6 kinase catalyzes the phosphorylation of a number of hepatic nonhistone nuclear proteins. This S6 kinase probably underlies the increased hepatic S6 phosphorylation observed after cycloheximide treatment, which in turn corresponds to the mitogen-activated S6 kinase.     

Phosphorylation of multiple proteins of both ribosomal subunits in rat cerebral cortex in vivo. Effect of adenosine 3'':5''-cyclic monophosphate.

Investigations were carried out on the phosphorylation of ribosomal proteins in vivo in cerebral cortices of immature rats. Two-dimensional electrophoresis revealed that the cerebral 40S subunit contained at least four ribosomal proteins which were phosphorylated in animals given [32P]orthophosphate intracisternally. These proteins exhibited electrophoretic properties similar to those of the constitutive basic proteins S2, S3a, S5 and S6. The cerebral 60S subunit contained several proteins that were phosphorylated in vivo, including three basic proteins with electrophoretic mobilities similar to those of ribosomal proteins L6, L14 and L19. Four other proteins associated with the 60S subunit that were more acidic were also phosphorylated. Phosphorylated congeners of 40S and 60S ribosomal proteins could often be detected in distinct protein-stained spots on two-dimensional electrophoretograms. The cerebral S6 protein consisted of at least five distinct species in different states of phosphorylation. Administration of N6O-2' dibutyryl cyclic AMP increased the proportion of the more phosphorylated congeners of the S6 protein, but appeared to have little or no effect on phosphorylation of other cerebral ribosomal proteins. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated S6-protein phosphorylation; N2O2'-dibutyryl cyclic GMP had no effect on this process. These observations indicate that several ribosomal proteins of both subunits are normally phosphorylated in rat cerebral cortex in situ. The results also suggest that selective and specific alterations in the phosphorylation state of the S6 ribosomal protein of the cerebral 40S subunit may accompany the production of cyclic AMP during neural activation.     

Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme.     

Rice serine/threonine kinase 1 is required for the stimulation of OsNug2 GTPase activity

Several GTPases are required for ribosome biogenesis and assembly. We recently identified rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), a YlqF/YawG family GTPase, as having a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating OsNug2 function, yeast two-hybrid screens were performed using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a candidate interacting protein. OsSTK1 appeared to interact with OsNug2 both in vitro and in vivo. OsSTK1 was found to have no effect on the GTP-binding activity of OsNug2; however, the presence of recombinant OsSTK1 in OsNug2 assay reaction mixtures increased OsNug2 GTPase activity. A kinase assay showed that OsSTK1 had weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Using yeast complementation testing, we identified a GAL::OsNug2(S209N) mutation-harboring yeast strain that exhibited a growth-defective phenotype on galactose medium at 39 °C, which was divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of OsNug2(S209N), which was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings indicate that OsSTK1 functions as a positive regulator of OsNug2 by enhancing OsNug2 GTPase activity. In addition, phosphorylation of OsNug2 serine 209 is essential for its complete function in biological functional pathway.