Evidence for a highly specific protein kinase phosphorylating two strongly acidic proteins of yeast 60 S ribosomal subunit

Kudlicki, Wiesław; Szyszka, Ryszard; Paleń, E; Gasior, E

doi:10.1016/0304-4165(80)90196-8

Cited by 36 publications

(19 citation statements)

References 20 publications

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“…We suspect that earlier descriptions of yeast CK1 published by others were plagued by proteolysis as well and led to confusing claims of molecular mass and to a profusion of molecular forms in the literature (Lerch et al, 1975;Kudlicki et al, 1980;Donella-Deana et al, 1985;Szyszka et al, 1985;Stembach and Kiintzel, 1987). Although no amino acid sequence data from these preparations are available, the amino acid composition of the 43-kDa form isolated by Lerch et al (1975) is clearly similar to that of the first n:380 amino acids of CKI1 (especially in Glu + Gln content).…”

Section: Discussionmentioning

confidence: 96%

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

Wang

Vančura

Mitcheson

et al. 1992

MBoC

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Two cDNAs encoding casein kinase-1 have been isolated from a yeast cDNA library and termed CKI1 and CKI2. Each clone encodes a protein of approximately 62,000 Da containing a highly conserved protein kinase domain surrounded by variable amino- and carboxy-terminal domains. The proteins also contain two conserved carboxy-terminal cysteine residues that comprise a consensus sequence for prenylation. Consistent with this posttranslational modification, cell fractionation experiments demonstrate that intact CKI1 is found exclusively in yeast cell membranes. Gene disruption experiments reveal that, although neither of the two CKI genes is essential by itself, at least one CKI gene is required for yeast cell viability. Spores deficient in both CKI1 and CKI2 fail to grow and, therefore, either fail to germinate or arrest as small cells before bud emergence. These results suggest that casein kinase-1, which is distributed widely in nature, plays a pivotal role in eukaryotic cell regulation.

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Section: Discussionmentioning

confidence: 96%

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

Wang

Vančura

Mitcheson

et al. 1992

MBoC

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“…Interestingly, there have been reports that the nonribosomal P-proteins are less phosphorylated than those on the ribosome (46,63) and that phosphorylation seems to increase the activities of P-proteins (32) or their affinities for the ribosome (46). P-proteins are phosphorylated by casein kinase type II, a cyclic AMP-independent kinase which can utilize either ATP or GTP (19,20,22,26,27,35). Serine residues in an acidic environment are generally good substrates for casein kinase type II, and in Artemia eL12 (P2) or eL12' (P1) the serine in the sequence .…”

Section: Discussionmentioning

confidence: 99%

Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

Rich¹,

Steitz²

1987

Mol. Cell. Biol.

273

153

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cDNA clones encoding three antigenically related human ribosomal phosphoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of LIO. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identities of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the PO, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.Acidic ribosomal proteins from diverse species have been studied extensively by a number of different techniques. These proteins, called A-proteins (acidic) or P-proteins (the phosphorylated eucaryotic A-proteins), are generally present in multiple copies on the ribosome and have isoelectric points in the range of pH 3 to 5, in contrast to most ribosomal proteins, which are single copy and basic. Aproteins have hydrophobic amino acid compositions (notably about 20% alanine) (34). Because of their distinct chemical properties, A-proteins can be easily dissociated from the ribosome by treatment with high salt and 50% ethanol; the A-proteins remain soluble whereas the remainder of the ribosomes or subunits precipitate (17). This procedure provides a uniform, simple protocol for the identification and isolation of A-proteins from many sources.The best characterized A-protein is the L7/L12 protein of Escherichia coli (L7 is the N-acetylated form of L12 [molecular weight, 12,164]), which has been localized to the stalk of the large (50S) ribosomal subunit (5,38). L7/L12 interacts with several translation factors, some of which bind and hydrolyze GTP, including initiation factor IF2, elongation factors EF-Tu and EF-G, and release factors RF1 and RF2 (5, 59). L7/L12 is required for binding of these factors as well as aminoacyl-tRNA to the ribosome (5, 59). Hydrodynamic and circular dichroism studies have suggested that L7/L12 and other A-proteins are prolate and have high alpha-helical contents (16). However, the crystal structure of the Cterminal domain of L7/L12 has been determined (28), and it is remarkably globular in light of the elongated shape of the whole molecule. The results of intraribosomal proteinprotein cross-linking studies and reconstitution experiments have depicted a complex...

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“…Earlier results from our laboratory have demonstrated the presence in the yeast cytosol of a specific protein kinase which phosphorylates the acidic proteins of a large ribosomal subunit [25]. A similar enzyme has also been observed in a protein fraction of a ribosomal salt wash [26].…”

mentioning

confidence: 72%

“…Unphosphorylated split proteins bind poorly and unspecifically to the core ribosomes deprived of the acidic proteins. Moreover, phosphorylated proteins restore the core particle capability to bind GDP in the presence of elongation factor 2 (EF-2) [4, 241. Earlier results from our laboratory have demonstrated the presence in the yeast cytosol of a specific protein kinase which phosphorylates the acidic proteins of a large ribosomal subunit [25]. A similar enzyme has also been observed in a protein fraction of a ribosomal salt wash [26].…”

mentioning

confidence: 72%

Specfic protein kinase from Saccharomyces cerevisiae cells phosphorylating 60S ribosomal proteins

Pilecki

Grankowski

Jacobs

et al. 1992

European Journal of Biochemistry

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A protein kinase, specific for 60s ribosomal proteins, has been isolated from Saccharomyces cerevisiae cells, purified to almost homogeneity and characterized. The isolated enzyme is not related to other known protein kinases. Enzyme purification comprised three chromatography steps ; DEAEcellulose, phosphocellulose and heparin -Sepharose. SDSjPAGE analysis of the purified enzyme, indicated a molecular mass of around 71 kDa for the stained single protein band. The specific activity of the protein kinase was directed towards the 60s ribosomal proteins L44, L44, L45 and a 38 kDa protein. All the proteins are phosphorylated only at the serine residues. None of the 40s ribosomal proteins were phosphorylated in the presence of the kinase. For that reason we have named the enzyme the 60s kinase. An analysis of the phosphopeptide maps of acidic ribosomal proteins, phosphorylated at either the 60s kinase or casein kinase 11, showed almost identical patterns. Using the immunoblotting technique, the presence of the kinase has been detected in extracts obtained from intensively growing cells. These findings suggest an important role played by the 60s kinase in the regulation of ribosomal activity during protein synthesis. 20 years ago, when Kaltschmidt and Wittmann described the gel electrophoresis procedure for the separation and identification of Escherichia coli ribosomal proteins [l], the two acidic proteins L7 and L12 (12 kDa) of the 50s subunit have become one of the most studied ribosomal proteins with respect to their three-dimensional structure and biological function.The functional and physicochemical equivalents of the procaryotic L7 and L12 are two ribosomal proteins, L44 and L45, from the eucaryotic 60s subunit (for review see [2]). Contrary to in animals, the ribosomes of Saccharomyces cerevisiae cells contain a third low-molecular-mass acidic protein. That protein, named L44', can be separated on DEAEcellulose [3] and by an electrofocusing procedure in acrylamide gel [4]. All three proteins have molecular masses of about 13 kDa, belong to 'split proteins ' [5] The phosphorylation of the crude eucaryotic ribosomes was initially reported by Kabat [lo] and by Loeb et al. [ll]. Later studies by other authors have shown that mammalian ribosomes are associated with cyclic-AMP-independent protein kinases [12-151. One of the enzymes has turned out to be a casein kinase type I1 (CKII) which phosphorylates proteins L44 and L45 [14]. For the yeast ribosomes, it has been found that the majority of phosphoproteins, y-32P labelled in vivo [16 -191 correspond to the proteins phosphorylated in vitro with . Two of them were identified as L44 and L45 [18, 19, 211 and are phosphorylated in vitro by the casein kinase type I1 [21, 221. Studies on the physiological role of the phosphorylation of ribosomal acidic proteins have been in progress for 10 years [23] and involved the yeast system. As shown in Ballesta's laboratory, the phosphorylation of L44, L44 and L45 might control the binding process of those proteins to ribosom...

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Evidence for a highly specific protein kinase phosphorylating two strongly acidic proteins of yeast 60 S ribosomal subunit

Cited by 36 publications

References 20 publications

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1.

Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

Specfic protein kinase from Saccharomyces cerevisiae cells phosphorylating 60S ribosomal proteins

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