Phosphorylation of the yeast ribosomal stalk. Functional effects and enzymes involved in the process

The ribosomal stalk is directly involved in the interaction of the elongation factors with the ribosome during protein synthesis. The stalk is formed by a complex of five proteins, four small acidic polypeptides and a larger protein which directly interacts with the rRNA at the GTPase center. In eukaryotes the acidic components correspond to the 12-kDa P1 and P2 proteins, and the RNA binding component is the P0 protein. All these proteins are found phosphorylated in eukaryotic organisms, and previous in vitro data suggested this modification was involved in the activity of this structure. Results from mutational studies have shown that phosphorylation takes place at a serine residue close to the carboxy end of the P proteins. Modification of this serine residue does not affect the formation of the stalk and the activity of the ribosome in standard conditions but induces an osmoregulation-related phenotype at 37 degrees C. The phosphorylatable serine is part of a consensus casein kinase II phosphorylation site. However, although CKII seems to be responsible for part of the stalk phosphorylation in vivo, it is probably not the only enzyme in the cell able to perform this modification. Five protein kinases, RAPI, RAPII and RAPIII, in addition to the previously reported CKII and PK60 kinases, are able to phosphorylate the stalk proteins. A comparison of the five enzymes shows differences among them that suggest some specificity regarding the phosphorylation of the four yeast acidic proteins. It has been found that some typical effectors of the PKC kinase stimulate the in vitro phosphorylation of the stalk proteins. All the data suggest that although phosphorylation is not involved in the interaction of the acidic P proteins with the ribosome, it can affect the ribosome activity and might participate in a possible ribosome regulatory mechanism.     

Ribosomal stalk protein phosphorylating activities in Saccharomyces cerevisiae

With ribosomal P protein as a substrate, five peaks of protein kinase activity are eluted after chromatography of a Saccharomyces cerevisiae cellular extract on DEAE-cellulose. Two of them correspond to CK-II and the other three have been called RAP-1, RAP-II, and RAP-III. RAP-I was previously characterized. RAP-III is present in a very small amount, which hindered its purification. RAP-II was further purified on phosphocellulose, heparin-Sepharose, and P protein-Sepharose, studied in detail, and compared with other acidic protein kinases, including RAP-I, CK-II, and PK60. RAP-II is shown by SDS-PAGE and centrifugation on glycerol linear density gradients to have a molecular mass of around 62 kDa and it is immunologically different from RAP-I and PK60. RAP-II phosphorylates the P proteins in the last serine residue at the highly conserved carboxyl terminal domain as other P-protein kinases. The ribosome-bound stalk P proteins are not equally phosphorylated by the different kinases. Thus, RAP-II and PK60 mainly phosphorylate P1beta and P2alpha whereas RAP-I and CK-II modify all of them. A comparative study of the K(m) and V(max) of the phosphorylation reaction by the different kinases using individual purified acidic proteins suggests changes in the substrate susceptibility upon binding to the ribosome. All the data available reveal clear differences in the characteristics of the various P protein kinases and suggest that the cell may use them to differentially modify the stalk depending, perhaps, on metabolic requirements.     

Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.     

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.     

The acidic protein binding site is partially hidden in the free Saccharomyces cerevisiae ribosomal stalk protein P0

The ribosomal stalk is essential for translation; however, its overall structure is poorly understood. Characterization of the region involved in the interactions between protein P0 and the 12 kDa acidic proteins P1 and P2 is fundamental to understand the assembly and function of this structure in the eukaryotic ribosome. The acidic protein content is important for the ribosome efficiency and affects the translation of specific mRNAs. By usage of a series of progressively truncated fragments of protein P0 in the two-hybrid test, a region between positions 213 and 250 was identified as the minimal protein part able to interact with the acidic proteins. Extensions at either end affect the binding capacity of the fragment either positively or negatively depending on the number of added amino acids. Deletions inside the binding region confirm its in vivo relevance since they drastically reduce the P0 interacting capacity with the 12 kDa acidic proteins, which are severely reduced in the ribosome when the truncated protein is expressed in the cell. Moreover, recombinant His-tagged P0 fragments containing the binding site and bound to Ni(2+)-NTA columns can form a complex with the P1 and P2 proteins, which is able to bind elongation factor EF2. The results indicate the existence of a region in P0 that specifically interacts with the acidic proteins. These interactions are, however, hindered by the presence of neighbor protein domains, suggesting the need for conformational changes in the complete P0 to allow the assembly of the ribosomal stalk.     

Mass spectrometry of ribosomes from Saccharomyces cerevisiae: implications for assembly of the stalk complex

The acidic ribosomal P proteins form a distinct protuberance on the 60 S subunit of eukaryotic ribosomes. In yeast this structure is composed of two heterodimers (P1alpha-P2beta and P1beta-P2alpha) attached to the ribosome via P0. Although for prokaryotic ribosomes the isolation of a pentameric stalk complex comprising the analogous proteins is well established, its observation has not been reported for eukaryotic ribosomes. We used mass spectrometry to examine the composition of the stalk proteins on ribosomes from Saccharomyces cerevisiae. The resulting mass spectra reveal a noncovalent complex of mass 77,291 +/- 7 Da assigned to the pentameric stalk. Tandem mass spectrometry confirms this assignment and is consistent with the location of the P2 proteins on the periphery of the stalk complex, shielding the P1 proteins, which in turn interact with P0. No other oligomers are observed, confirming the specificity of the pentameric complex. At lower m/z values the spectra are dominated by individual proteins, largely from the stalk complex, giving rise to many overlapping peaks. To define the composition of the stalk proteins in detail we compared spectra of ribosomes from strains in which genes encoding either or both of the interacting stalk proteins P1alpha or P2beta are deleted. This enables us to define novel post-translational modifications at very low levels, including a population of P2alpha molecules with both phosphorylation and trimethylation. The deletion mutants also reveal interactions within the heterodimers, specifically that the absence of P1alpha or P2beta destabilizes binding of the partner protein on the ribosome. This implies that assembly of the stalk complex is not governed solely by interactions with P0 but is a cooperative process involving binding to partner proteins for additional stability on the ribosome.     

Tag-mediated fractionation of yeast ribosome populations proves the monomeric organization of the eukaryotic ribosomal stalk structure

The analysis of the not well understood composition of the stalk, a key ribosomal structure, in eukaryotes having multiple 12 kDa P1/P2 acidic protein components has been approached using these proteins tagged with a histidine tail at the C-terminus. Tagged Saccharomyces cerevisiae ribosomes, which contain two P1 proteins (P1 alpha and P1 beta) and two P2 proteins (P2 alpha and P2 beta), were fractionated by affinity chromatography and their stalk composition was determined. Different yeast strains expressing one or two tagged proteins and containing either a complete or a defective stalk were used. No indication of protein dimers was found in the tested strains. The results are only compatible with a stalk structure containing a single copy of each one of the four 12 kDa proteins per ribosome. Ribosomes having an incomplete stalk are found in wild-type cells. When one of the four proteins is missing, the ribosomes do not carry the three remaining proteins simultaneously, containing only two of them distributed in pairs made of one P1 and one P2. Ribosomes can carry two, one or no acidic protein pairs. The P1 alpha/P2 beta and P1beta/P2 alpha pairs are preferentially found in the ribosome, but they are not essential either for stalk assembly or function.     

Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses

Trichosanthin (TCS) is a type I ribosome-inactivating protein that inactivates ribosome by enzymatically depurinating the A⁴³²⁴ at the α-sarcin/ricin loop of 28S rRNA. We have shown in this and previous studies that TCS interacts with human acidic ribosomal proteins P0, P1 and P2, which constitute the lateral stalk of eukaryotic ribosome. Deletion mutagenesis showed that TCS interacts with the C-terminal tail of P2, the sequences of which are conserved in P0, P1 and P2. The P2-binding site on TCS was mapped to the C-terminal domain by chemical shift perturbation experiments. Scanning charge-to-alanine mutagenesis has shown that K173, R174 and K177 in the C-terminal domain of TCS are involved in interacting with the P2, presumably through forming charge–charge interactions to the conserved DDD motif at the C-terminal tail of P2. A triple-alanine variant K173A/R174A/K177A of TCS, which fails to bind P2 and ribosomal stalk in vitro, was found to be 18-fold less active in inhibiting translation in rabbit reticulocyte lysate, suggesting that interaction with P-proteins is required for full activity of TCS. In an analogy to the role of stalk proteins in binding elongation factors, we propose that interaction with acidic ribosomal stalk proteins help TCS to locate its RNA substrate.     

Role of the ribosomal stalk components in the resistance of Aspergillus fumigatus to the sordarin antifungals

Aspergillus fumigatus, an important human nosocomial pathogen, is resistant to sordarin derivatives, a new family of antifungals that inhibit protein synthesis by interaction with the EF-2-ribosomal stalk complex. To explore the role of the A. fumigatus ribosome in the resistance mechanism, the fungal stalk proteins were biochemically and genetically characterized and expressed in the sensitive Saccharomyces cerevisiae. Two acidic phosphoproteins homologous to the 12 kDa P1 and P2 proteins described in other organisms were found together with the 34 kDa P0 protein, the third stalk component. The genes encoding each fungal stalk protein were expressed in mutant S. cerevisiae strains lacking the equivalent proteins. Both AfP1 and AfP2 proteins interact with their yeast counterparts of the opposite type and bind to the ribosomal particles in the presence of either the S. cerevisiae or the A. fumigatus P0 protein. The A. fumigatus acidic phosphoproteins did not alter the yeast ribosome sordarin sensitivity. On the contrary, the presence of the fungal P0 induces in vivo and in vitro resistance to sordarin derivatives when present in the yeast ribosome. The mutations A117-->E, P122-->R and G124-->V in A. fumigatus P0 reduce the resistance capacity of the protein. An S. cerevisiae strain with the complete ribosomal stalk of A. fumigatus was obtained, which could be useful for the screening of new antifungals against this pathogenic fungus.     

Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum

Phosphorylation of acidic ribosomal proteins P1/P2-P0 is a common phenomenon in eukaryotic organisms. It was found previously that in Trichosporon cutaneum, unlike in other yeast species, in addition to the two acidic ribosomal proteins, two other proteins of 15 kDa and 19 kDa of the small ribosomal subunit were phosphorylated. Here we describe two protein kinases: CKI and CKII, which are engaged in the modification of T. cutaneum ribosomal proteins. The acidic ribosomal proteins and the protein of 19 kDa were modified by CKII associated with ribosomes, while the protein of 15 kDa was modified by CKI. Protein kinase CKI was purified from cell-free extract (CKIC) and from ribosomal fraction (CKIR). The molecular mass of CKIC was established at 33 kDa while that of CKIR at 35-37 kDa. A protein of 40 kDa copurified with CKIR but not CKIC. Heparin significantly increased 40 kDa protein phosphorylation level by CKIR. Microsequencing analysis revealed the presence of CKI recognition motifs in the N-terminal fragment of the 40 kDa protein.     

Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components

The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.     

Structural and functional characterization of the amino terminal domain of the yeast ribosomal stalk P1 and P2 proteins

The essential ribosomal stalk is formed in eukaryotes by a pentamer of two P1–P2 protein heterodimers and the P0 rRNA binding protein. In contrast to the highly stable prokaryotic complex, the P1 and P2 proteins in the eukaryotic stalk undergo a cyclic process of assembly and disassembly during translation that seems to modulate the ribosome activity. To better understand this process, the regions of the Saccharomyces cerevisiae P1α and P2β proteins that are directly involved in heterodimer formation and ribosome binding have been characterized using a series of P1α/P2β chimeras. The region required for a stable interaction with the ribosome is formed by the first three predicted α-helices in the N-terminal domain of both proteins. The same region is required for heterodimer formation in P2β but the third helix is dispensable for this association in P1α. It seems, therefore, that stable ribosome binding is more structurally demanding than heterodimerization. A fourth predicted α-helix in the N-terminal domain of P1α and P2β appears not to be involved in the assembly process but rather, it contributes to the conformation of the proteins by apparently restricting the mobility of their C-terminal domain and paradoxically, by reducing their activity. In addition, the study of P1/P2 chimeras showed that the C-terminal domains of these two types of protein are functionally identical and that their protein specificity is exclusively determined by their N-terminal domains.     

Molecular behavior of human Mrt4 protein,MRTO4, in stress conditions is regulated by its C-terminal region

Protein Mrt4 is one of trans-acting factors involved in ribosome biogenesis, which in higher eukaryotic cells contains a C-terminal extension similar to the C-terminal part of ribosomal P proteins. We show that human Mrt4 (hMrt4/MRTO4) undergoes phosphorylation in vivo and that serines S229, S233, and S235, placed within its acidic C-termini, have been phosphorylated by CK2 kinase in vitro. Such modification does not alter the subcellular distribution of hMrt4 in standard conditions but affects its molecular behavior during ActD induced nucleolar stress. Thus, we propose a new regulatory element important for the stress response pathway connecting ribosome biogenesis with cellular metabolism.     

Phosphorylation by the varicella-zoster virus ORF47 protein serine kinase determines whether endocytosed viral gE traffics to the trans-Golgi network or recycles to the cell membrane

Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as "team players" in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.     

The E7 protein of human papillomavirus type 16 is phosphorylated by casein kinase II

The E7 protein of human papillomavirus type 16 (HPV16) transforms cultured cells and cooperates with the ras or fos oncogenes in the transformation of primary cells. In this study we have investigated the phosphorylation of E7. When we immunoprecipitated E7 from CaSki cells with a rabbit polyclonal antiserum to a bacterial fusion protein (trpE-E7), we found that E7 was phosphorylated at serine residues contained in five characteristic thermolysin peptides. Immunoprecipitated E7, and fusion proteins harboring the E7 protein from various HPV types, could all be specifically phosphorylated in vitro by the ubiquitous, growth factor-activated casein kinase II (CKII). Comparative peptide mapping showed that the sites of in vivo and in vitro phosphorylation are the same. CKII was shown previously to specifically phosphorylate serine or threonine residues within a cluster of acidic amino acids. The E7 protein contains such a sequence between amino acids 30 and 37. When a synthetic peptide corresponding to this region of E7 was phosphorylated by CKII in vitro, its thermolysin digestion products were the same as those in the phosphorylated E7 protein. We conclude that E7 is phosphorylated in vivo only at serines within the predicted CKII site and that CKII, or a CKII-like enzyme, participates in the reaction. Both the E1A and SV40 large T proteins contain similar CKII consensus sites proximal to the regions required for their associations with the retinoblastoma gene product (p105Rb). Thus it is conceivable that CKII phosphorylation can modulate the interaction between the transforming proteins and the retinoblastoma gene product.     

Functional characterization of the major and minor phosphorylation sites of the P protein of Borna disease virus

The phosphoprotein P of Borna disease virus (BDV) is an essential cofactor of the viral RNA-dependent RNA polymerase. It is preferentially phosphorylated at serine residues 26 and 28 by protein kinase C epsilon (PKCepsilon) and, to a lesser extent, at serine residues 70 and 86 by casein kinase II (CKII). To determine whether P phosphorylation is required for viral polymerase activity, we generated P mutants lacking either the PKCepsilon or the CKII phosphate acceptor sites by replacing the corresponding serine residues with alanine (A). Alternatively, these sites were replaced by aspartic acid (D) to mimic phosphorylation. Functional characterization of the various mutants in the BDV minireplicon assay revealed that D substitutions at the CKII sites inhibited the polymerase-supporting activity of P, while A substitutions maintained wild-type activity. Likewise, D substitutions at the PKC sites did not impair the cofactor function of BDV-P, whereas A substitutions at these sites led to increased activity. Interestingly, recombinant viruses could be rescued only when P mutants with modified PKCepsilon sites were used but not when both CKII sites were altered. PKCepsilon mutant viruses showed a reduced capacity to spread in cell culture, while viral RNA and protein expression levels in persistently infected cells were almost normal. Further mutational analyses revealed that substitutions at individual CKII sites were, with the exception of a substitution of A for S86, detrimental for viral rescue. These data demonstrate that, in contrast to other viral P proteins, the cofactor activity of BDV-P is negatively regulated by phosphorylation.     

A serine residue in the N-terminal acidic region of rat RPB6, one of the common subunits of RNA polymerases, is exclusively phosphorylated by casein kinase II in vitro.

RPB6 is one of the common subunits of all eukaryotic RNA polymerases and is indispensable for the enzyme function. Here, we isolated a rat cDNA encoding RPB6. It contained 127 amino acid (a.a.) residues. From alignment of RPB6 homologues of various eukaryotes, we defined two conserved regions, i.e. an N-terminal acidic region and a C-terminal core. In this study, we investigated in vitro phosphorylation of rat RPB6 by casein kinase II (CKII), a pleiotropic regulator of numerous cellular proteins. Three putative CKII-phosphorylated a.a. within rat RPB6 were assigned. We found that serines were phosphorylated by CKII in vitro. Mutagenesis studies provided evidence that a serine at a.a. position 2 was exclusively phosphorylated. Finally, an RPB6-engaged in-gel kinase assay clarified that CKII was a prominent protein kinase in rat liver nuclear extract that phosphorylates RPB6. Therefore, RPB6 was implied to be phosphorylated by CKII in the nucleus. We postulate that the N-terminal acidic region of the RPB6 subunit has some phosphorylation-coupled regulatory functions.     

Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis

The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1α, P1β, P2α, and P2β. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1α, P1β, P2α, and P2β. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.