Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA

Shimizu, Takeo; Nakagaki, Masayuki; Nishi, Yoshinori; Kobayashi, Yuji; Hachimori, Akira; Uchiumi, Toshio

doi:10.1093/nar/gkf379

Cited by 69 publications

(95 citation statements)

References 47 publications

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“…It is well known that the C-terminal 120 amino-acid residues of P0 is responsible for the interaction with elongation factor EF-2 and P1/P2 proteins (Uchiumi and Kominami, 1997), whereas an arginine-rich region (residues 44-67 in the rat protein) in the N-terminal half of P0 has been found to be responsible for binding of the pentamer to RNA (Shimizu et al, 2002). This argininerich region is 100% conserved among the frog, rat, chicken and human proteins (Wu and Storey, 2005).…”

Section: Discussionmentioning

confidence: 99%

Ribosomal phosphoprotein P0 interacts with GCIP and overexpression of P0 is associated with cellular proliferation in breast and liver carcinoma cells

et al. 2007

Oncogene

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The ribosomal acidic P0 protein, an essential component of the eukaryotic ribosomal stalk, was found to interact with the helix-loop-helix protein human Grap2 and cyclin D interacting protein (GCIP)/D-type cyclin-interacting protein 1/human homolog of MAID protein. Using in vivo and in vitro binding assays, we show that P0 can interact with the N and C termini of GCIP via its N-terminal 39-114 amino-acid residues. Although the P0-GCIP complex was detected mainly in cytoplasmic fraction, polysome profile analysis indicated that the P0-GCIP complex did not coelute with either polysomes or 60S ribosomes, suggesting that GCIP associates with the free form of P0 in the cytoplasm. Transfection of GCIP into MCF-7 cells resulted in decreased levels of pRb phosphorylation. Cotransfection of P0 with GCIP, however, resulted in GCIP-mediated reduction of pRb phosphorylation level which was repressed by P0. Furthermore, overexpression of P0 in breast cancer and hepatocellular cancer cell lines promoted cell growth and colony formation compared to control transfectants. Overexpression of P0 also increased cyclin D1 expression and phosphorylation of pRb at Ser780. Interestingly, P0 mRNA was overexpressed in 12 of 20 pairs of breast cancer/ normal breast specimens (60%). Together, these data indicate that P0 overexpression may cause tumorigenesis in breast and liver tissues at least in part by inhibiting GCIP-mediated tumor suppression.

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Section: Discussionmentioning

confidence: 99%

Ribosomal phosphoprotein P0 interacts with GCIP and overexpression of P0 is associated with cellular proliferation in breast and liver carcinoma cells

et al. 2007

Oncogene

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“…For aEF-1α, HiTrap SP-sepharose was used instead of HiTrap Q-sepharose. Silkworm P0, P1, and P2 proteins and their variants were purified as described by Shimizu et al (35). The silkworm stalk complex was reconstituted by mixing these components (11).…”

Section: Methodsmentioning

confidence: 99%

“…The silkworm stalk complex was used to replace the E. coli L10-L12 complex in the bacterial ribosome, and the GTPase activity that depended on eukaryotic eEF-2 was assayed, as described by Shimizu et al (35).…”

Section: Methodsmentioning

confidence: 99%

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

Nomura

Honda

Baba

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1α, but not aIF2γ, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0ðaP1Þ 2 ðaP1Þ 2 ðaP1Þ 2 , can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency.GTPase-associated center | ribosome protein P0 | ribosome protein P1 | hyperthermophilic archaeon M ajor dynamic steps in translational initiation, elongation, and termination are promoted by the actions of several translational GTPase factors (1-3). During the elongation step, two elongation factors bind alternately to the ribosome in their GTP-bound states. After they have carried out their respective functions, which are linked to GTP hydrolysis, they then dissociate from the ribosome. The large ribosomal subunit contains an active center, termed the GTPase-associated center or factorbinding center, which interacts with the elongation factors (4). The GTPase-associated center plays a crucial role in the recruitment of translation factors, GTP hydrolysis, and the release of inorganic phosphate, which is required for the subsequent dissociation of the factor. Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5-9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).In the case of bacteria, the structure and function of the stalk protein L7/L12 (termed L12 hereafter) is well established. The L12 protein is composed of an N-terminal dimerization domain and a globular C-terminal doma...

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“…There is also a consensus for an anchorage of the P1/P2 dimer by a direct interaction of the P1 N-terminal domain, but not the P2 N-terminal domain with P0 (Gonzalo et al, 2001;Shimizu et al, 2002;Zurdo et al, 2000).…”

Section: Binding Of P1/p2 Dimers (And Equivalents) To P0 (And Equivalmentioning

confidence: 99%

The puzzling lateral flexible stalk of the ribosome

Gonzalo

Reboud

2003

Biology of the Cell

127

131

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The lateral flexible stalk of the large ribosomal subunit is made of several interacting proteins anchored to a conserved region of the 28S (26S) rRNA termed the GTPase-associated domain or thiostrepton loop. This structure is demonstrated to adopt puzzling changes of conformation following the different steps of the elongation cycle. Some of these proteins termed the P-proteins in eukaryotes and L10 and L7/L12 in bacteria, present little structural similarities between Eubacteria on one side and Archae and Eukaryotes on the other side. However, up to now, these proteins seem to present a similar macromolecular organisation and they have been involved in the same functions. Convincing evidence attests that these proteins participate in elongation factor binding to the ribosome, and it has been suggested that these proteins might be evolved in a GTP hydrolysis activating protein activity. Involvement of these proteins in the translational mechanism is discussed. Moreover, in eukaryotes, small P-proteins are also found as isolated proteins in a cytoplasmic pool that exchanges with the ribosome-associated P-proteins. Moreover, a part of the ribosomal proteins is phosphorylated (hence their P-protein names). The biological signification of these particularities is discussed.

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Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA

Cited by 69 publications

References 47 publications

Ribosomal phosphoprotein P0 interacts with GCIP and overexpression of P0 is associated with cellular proliferation in breast and liver carcinoma cells

Ribosomal phosphoprotein P0 interacts with GCIP and overexpression of P0 is associated with cellular proliferation in breast and liver carcinoma cells

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

The puzzling lateral flexible stalk of the ribosome

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