Oligomerization properties of the acidic ribosomal P-proteins from Saccharomyces cerevisiae: effect of P1A protein phosphorylation on the formation of the P1A-P2B hetero-complex.

Acidic ribosomal P-proteins form, in all eukaryotic cells, a lateral protuberance, the so-called 'stalk', which is directly involved in translational activity of the ribosomes. In Saccharomyces cerevisiae cells, there are four distinct P-proteins: P1A, P1B, P2A and P2B. In spite of the high level of their structural homology, they are not completely equivalent and may perform different functions. As yet, the protein-protein interactions between yeast P-proteins have not been fully defined. In this paper, the interplay between yeast P-proteins has been investigated by means of a two-hybrid system, chemical cross-linking and gel filtration. The data presented herein show that all P-proteins are able to form homo-oligomeric complexes. By analyzing hetero-interactions, we were able to detect strong interactions between P1A and P2B proteins. Additionally, the pair of P1B and P2A proteins is also able to form a hetero-complex, though at a very low efficiency. All P-proteins are phosphorylated by numerous protein kinases. Using the multifunctional protein kinase CK II, we have shown that incorporation of phosphate into P1A protein can exert its effect on the hetero-oligomerization process, namely by preventing the formation of the hetero-oligomer P1A-P/P2B. These findings are the first to show differences in the oligomerization behavior of the yeast P-proteins; moreover, they emphasize a significant impact of the phosphorylation on the formations of P-protein complex.

Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells.

Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein substrate not only depends on the structure of the polypeptide chain around the target amino acid but also on its native structure within the 80S ribosome.

A minor species of a type I casein kinase from yeast phosphorylating threonine residues of protein substrate.

Protein kinase of Mr 23 000 was isolated from yeast and purified to apparent homogeneity. The enzyme preferentially phosphorylated casein and phosvitin in the presence of ATP as a phosphoryl donor. Its activity was neither affected by cyclic nucleotides nor by heparin. The kinase displayed practically the same substrate specificity as a typical casein kinase I from yeast (Kudlicki, W., Szyszka, R., Palen, E. and Gasior, E. (1980) Biochim. Biophys. Acta 633, 376-385) except that it phosphorylated threonine instead of serine residues in protein substrates.

A type-1 casein kinase from yeast phosphorylates both serine and threonine residues of casein. Identification of the phosphorylation sites.

A protein kinase (casein kinase 1A) active on casein and phosvitin but not on histones has been purified to near homogeneity from yeast cytosol and meets most criteria for being considered a type-1 casein kinase: it is a monomeric enzyme exhibiting an Mr of about 27 kDa by sucrose gradient centrifugation: it is not affected by inhibitors of type-2 casein kinases, such as heparin and polyglutamate, and shows negligible affinity for GTP. It also readily phosphorylates the residue Ser-22 of beta-casein located within the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser22-Ile-Thr-Arg- which is typically affected by casein kinases of the first class. On the other hand, casein kinase 1A displays the unusual property of phosphorylating threonine residue(s) in both whole casein and alpha s1-casein. The threonine residue phosphorylated in alpha s1-casein and accounting for most of the 32P incorporated into this protein by casein kinase 1A has been identified as Thr-49, which occurs in the sequence -Ser(P)-Glu-Ser(P)-Thr(P*)49-Glu-Asp-Gln-, whose two Ser(P) residues are already phosphorylated in the native protein. It is concluded that some type-1 casein kinases can also phosphorylate threonine residues provided they fulfil definite structural requirements, probably an acidic cluster near their N-terminal side.

Endogenous inhibitor of a cyclic AMP-independent (A type) protein kinase.

The cytosol fraction of the cells of Saccharomyces cerevisiae contains a low-molecular-mass, heat-stable inhibitor for endogenous cAMP-independent protein kinase. The inhibitor (Mr 15 000) is specific toward the protein kinase of A type, while the protein kinase of G type and the catalytic subunit of cAMP-dependent protein kinase are not affected. The following results suggest a protein structure of the inhibitor: 1. preincubation of the inhibitor with trypsin totally abolished its activity; 2. the inhibitor can be labelled by reductive alkylation of the amino acids with [14C]formaldehyde and sodium cyanoborohydride. The kinetic experiments have shown that the inhibitor is a competitive effector of the protein kinase of A type with respect to the protein substrate.

An in vivo and in vitro phosphorylation of yeast ribosomal proteins.

Phosphorylation of yeast ribosomal proteins has been demonstrated in vivo and in vitro. 32-P-labelled product represents an ester-linked class of phosphoprotein. Acrylamide-gel electrophoresis has shown that in both types of experiments radioactive proteins migrate similarly; this might indicate that closely related groups of proteins become phosphorylated in vivo and in vitro. In the presence of [32-P] ATP the amount of covalently bound phosphate was 1.0 - 1.2 moles/mole of ribosome. The phosphorylation of ribosomal proteins did not appreciably affect the activity of ribosomes in a cell-free protein-synthesizing system containing poly(U) and elongation factors.

Immunological properties and peptide mapping of two type I casein kinases from yeast.

Two protein kinases of Mr 43,000 and 23,000 from yeast, belonging to type-1 casein kinases, were purified to apparent homogeneity and used for investigation of their immunological affinity and for comparison of their peptide map patterns. The results obtained showed that antibodies against the 43 kDa kinase did not react with the 23 kDa enzyme. Moreover, the peptide maps of the radioiodinated kinases obtained either by chemical cleavage of peptide bonds in the presence of CNBr or by a limited digestion with V8 protease were completely different. All these observations point to the lack of relatedness between the two investigated enzymes.

The phosphorylation sites of ribosomal P proteins from Saccharomyces cerevisiae cells by endogenous CK-2, PK60S and RAP protein kinases.

The phosphorylation sites of ribosomal acidic proteins (P proteins) from Saccharomyces cerevisiae were studied in vivo and in vitro by using CK-2, PK60S and RAP protein kinases. The three enzymes phosphorylate the last serine residues located in a highly conserved carboxyl end of the polypeptide chains. This was established by two-dimensional analysis of tryptic phosphopeptides from 32P-labelled proteins YP1 alpha, YP1 beta, YP2 alpha and YP2 beta, and by kinetic studies of the protein kinases with synthetic peptides corresponding to the fragments of endogenous ribosomal acidic polypeptides. In experiments with both endogenous P proteins and synthetic peptides as substrates protein kinase PK60S demonstrated unusual substrate specificity. In contrast to CK-2 and RAP protein kinases, PK60S phosphorylates predominantly two of the four P proteins, YP1 alpha and YP2 beta, with kinetic constants dependent on the primary structure of the N-terminal region of the polypeptide containing the target residue. The neutral amino acid, alanine, at position 3 in the peptide AAEESDDD (polypeptide fragments of YP1 beta and YP2 alpha) decreases the K(m) value more than 10-fold by comparison with the basic lysine residue at the same position in the peptide AKEESDDD (polypeptide fragments of YP1 alpha and YP2 beta).

Extraribosomal function of the acidic ribosomal P1-protein YP1alpha from Saccharomyces cerevisiae.

The yeast acidic ribosomal P-proteins YP1alpha, YP1beta, YP2alpha and YP2beta were studied for a possible transactivation potential beside their ribosomal function. The fusions of P-proteins with the GAL4 DNA-binding domain were assayed toward their transcriptional activity with the aid of reporter genes in yeast. Two of the P-proteins, YP1alpha and YP1beta, exhibited transactivation potential, however, only YP1alpha can be regarded as a potent transactivator. This protein was able to transactivate a reporter gene associated with two distinct promoter systems, GAL1 or CYC1. Additionally, truncated proteins of YP1alpha and YP1beta were analyzed. The N-terminal part of YP1alpha fused to GAL4-BD showed transactivation potential but the C-terminal part did not. Our results suggest a putative extraribosomal function for these ribosomal proteins which consequently may be classified as "moonlighting" proteins.

Halogenated benzimidazole inhibitors of phosphorylation, in vitro and in vivo, of the surface acidic proteins of the yeast ribosomal 60S subunit by endogenous protein kinases CK-II and PK60S.

Several halogeno benzimidazoles and 2-azabenzimidazoles, previously shown to be relatively selective inhibitors of protein kinases CK-I and/or CK-II from various sources, including CK-II from yeast [Szyszka et al. (1995) Biochem. Biophys. Res. Commun. 208, 418-424] inhibit also the yeast ribosomal protein kinase PK60S. The most effective inhibitor of CK-II and PK60S was tetrabromo-2-azabenzimidazole ](TetraBr-2-azaBz), which was competitive with respect to ATP (and GTP in the case of CK-II) with Ki values of 0.7 microM for CK-II, and 0.1 microM for PK60S PK60S phosphorylates only three (YP1 beta', YP2 alpha) out of five polypeptides of pp13 kDa acidic proteins of 60S subunit phosphorylated by CK-II [Szyszka et al. (1995) Acta Biochim. Polon. 42, 357-362]. Accordingly, TetraBr-2-azaBz inhibits phosphorylation only of these polypeptides, catalysed by PK60S . Addition of TetraBr-2-azaBz to cultures of yeast cells, at concentrations which were without effect on cell growth, led to inhibition of intracellular phosphorylation of ribosomal acidic proteins, paralleling that observed in vitro. TetraBr-2-azaBz is shown to be a useful tool for studies on the intracellular regulation of phosphorylation of the ribosomal 60S acidic proteins, which are involved in formation of active ribosomes.

On the role of cyclic AMP-independent protein kinases in the modification of yeast ribosomal proteins in vivo.

Two proteins of yeast 40S ribosome subunit and four proteins of the 60S ribosome subunit were labelled in vivo with [32P]orthophosphate. Five of these proteins were phosphorylated by protein kinase 3, an enzyme which is cyclic AMP-independent and uses ATP and GTP as phosphoryl donors. Two proteins, belonging to the 60S ribosome subunit were phosphorylated by another, highly specific, cyclic AMP-independent protein kinase 1 B. Both in vivo and in vitro the most extensively phosphorylated protein species were acidic proteins, L44, L45 (according to the nomenclature of Kruiswijk & Planta, Molec. Biol. Rep., 1, 409-415, 1974) possibly corresponding to bacterial L7 and L12 proteins. The 40S ribosomal protein, S9, analogous to mammalian S6 protein, was phosphorylated in vivo but was not phosphorylated in vitro by either of the cyclic AMP-independent protein kinases. The obtained results clearly indicate that cyclic AMP-independent yeast protein kinases might be involved in the modification in vivo of some ribosomal proteins, in particular of the strongly acidic proteins of 60S ribosome subunit.

Proteins of yeast ribosomal subunits: number and general properties.

1. Saccharomyces cerevisiae at the early stationary phase of growth accumulate 80 S ribosomes, easily dissociating into subunits, which retain full activity in phenylalanine polymerization in vitro. A simplified and efficient technique for large-scale preparation of yeast ribosomal subunits is proposed. 2. Presence of 34 proteins in 40 S subunit and 42 proteins in 60 S subunit was demonstrated by two-dimensional acrylamide-gel electrophoresis. Both ribosomal subunits contain acidic proteins: three in 60 S and six or seven in 40 S subunit. It seems that two of them correspond to prokaryotic proteins, L7 and L12. The total number of yeast ribosomal proteins is similar to those obtained for other Eukaryota.

Further studies on the quaternary structure of yeast casein kinase II.

Casein kinase type II were isolated by the same procedure, from rat liver, human placenta, Querin carcinoma and yeast, and characterized. The mammalian enzymes were composed of three subunits alpha, alpha' and beta, whereas yeast kinase was composed of two subunits alpha and alpha'. It was shown that the catalytic activity, substrate and phosphate donor specificity, sensitivity to heparin and spermine were the same for all the kinases tested. The results give additional support to the suggestion [1] that the beta subunit is not required for optimal activity and specificity of yeast casein kinase II. The quaternary structure of the yeast enzyme of a molecular weight of approximately 150 000 is proposed as alpha2 alpha'2.

Differential phosphorylation of ribosomal acidic proteins from yeast cell by two endogenous protein kinases: casein kinase-2 and 60S kinase.

The native 80S ribosomes isolated from Saccharomyces cerevisiae (strain W303) cells was phosphorylated by two endogenous protein kinases: multifunctional casein kinase-2 (CK-2) and specific 60S kinase. Three acidic proteins within the 60S ribosomal subunit: YP1 beta, YP1 beta' and YP2 alpha are phosphorylated by both kinases. The other two proteins: YP1 alpha and YP2 beta are predominantly phosphorylated by CK-2 but not by 60S kinase. This was confirmed in the experiment with the recombinant protein, YP2 beta, as a substrate, which is practically not phosphorylated by specific 60S kinase. These results together with the previous data based on the target amino-acid sequences suggest that, in addition to the multifunctional casein kinase-2 and specific 60S kinase, there exist probably other protein kinase(s) which phosphorylate the ribosomal acidic proteins in the cell.

No effect of cAMP on protein synthesis in reticulocyte lysates.

The results of a series of experiments are interpreted to indicate that protein synthesis in reticulocyte lysates is not affected by the reticulocyte cAMP-dependent protein kinase. The catalytic subunit of this enzyme was isolated to apparent homogeneity. Also, the protein inhibitor of this protein kinase was isolated from muscle. Neither physiological concentrations of cAMP nor any of these protein components had a detectable effect on protein synthesis in reticulocyte lysates in the presence or absence of exogenous heme. Phosphorylation of the smallest subunit of eukaryotic initiation factor 2 or the 90,000 to 100,000-dalton peptide associated with eukaryotic initiation factor 2 kinase activity were not affected by the activity of the cAMP-dependent protein kinase under conditions in which exogenous heme has a pronounced effect on these reactions.

Isolation and properties of two protein kinases from yeast which phosphorylate casein and some ribosomal proteins.

Three fractions of protein kinase from postribosomal supernatant of Saccharomyces cerevisiae, active in phosphorylation of casein, were resolved on DEAE-cellulose. Two of these fractions: protein kinase 1 and protein kinase 3, were further purified about 1000 and 1800-fold respectively. The kinase 1 appeared to exist as a monomer with a molecular weight of 50 000 and utilized only ATP as phosphoryl donor. The protein kinase 3 was an aggregated form of enzyme with a molecular weight of above half a million and used both ATP and GTP for protein phosphorylation. Both isolated enzymes showed variations in respect to Michaelis constants, and inhibitory effects exerted by monovalent cations and nucleotide phosphates. The activity of the kinases was not affected by the presence of cAMP (adenosine 3':5'-monophosphate) or cGMP, however, only protein kinase 1 appeared to be a cAMP nucleotide-independent enzyme. Despite these differences both enzymes equally phosphorylated two strongly acidic proteins of the 60-S ribosome subunit, possibly related to L7, L12 of Escherichia coli.

Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast.

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized. It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 X 10(-6)M and 5.5 X 10(-5)M, respectively. Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [gamma-32P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.