Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit

Tohgo, Akira; Takasawa, Shin; Munakata, Hiroshi; Yonekura, Hideto; Hayashi, Norio; Okamoto, Hiroshi

doi:10.1016/0014-5793(94)80188-6

Cited by 38 publications

(29 citation statements)

References 23 publications

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“…The screening of human cDNA libraries using antibodies directed against the purified protein enabled the isolation of a full-length cDNA encoding a protein with a calculated molecular mass of 32 kDa and an apparent molecular weight of 37 kDa, after in vitro translation of hybrid-selected mRNA and SDS-PAGE analysis [4]. Highly homologous cDNAs were subsequently isolated from human colon cancer cell lines [5] and obtained in a study on the structure and sequence determination of the rat 40S ribosomal subunit [6]. LPB-p40 was localized on 40S ribosomes [7] and in the nucleus [8], while 67LR was located on the cell surface, where, in addition to its role as a high affinity laminin receptor, it was shown to function as the receptor for elastin [9] and as a positional marker for the differentiation of the fetal eye organ [10].…”

Section: Introductionmentioning

confidence: 99%

siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis

Susantad

Smith

2008

Cellular and Molecular Biology Letters

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Abbreviations used: GAPDH -glyceraldehyde 3-phosphate dehydrogenase; LBP-p40 -laminin-binding protein precursor p40; LRP -laminin receptor precursor; PrP c -cellular prion protein; siRNA -small interfering RNAs; 37LBP -37-kDa laminin binding protein; 67LR -67-kDa laminin receptor However, few studies have investigated the cellular consequence of the ablation of this gene's expression. To address this issue, the expression of the 37/67-kDa high affinity laminin receptor was knocked out with several siRNA constructs via RNA interference in transformed liver (Hep3B) cells. In each case where the message was specifically ablated, apoptosis was induced, as determined by annexin V/propidium iodide staining, and by double staining with annexin V and an antibody directed against the 37/67-kDa high affinity laminin receptor. These results suggest that this protein plays a critical role in maintaining cell viability.

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Section: Introductionmentioning

confidence: 99%

siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis

Susantad

Smith

2008

Cellular and Molecular Biology Letters

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“…It has been suggested that LBP-p40 is required for translation in plants ( GarciaHernandez et al, 1994), lower eukaryotes (Rosenthal and Wordeman, 1995;Demianova et al, 1996) and presumably in higher eukaryotes (Auth and Brawerman, 1992;Tohgo et al, 1994). The rate of protein synthesis in AS-p40 clones was assayed by 3 H-leucine incorporation.…”

Section: Discussionmentioning

confidence: 99%

“…However, the nomenclature of the laminin-binding protein precursor generated some confusion with progress in the analysis of p40 since LBP-p40 appeared to exhibit multiple functions in various aspects of cell growth, embryonic development, and cancer progression. Several lines of evidence have indicated that the p40 is localized exclusively in the cytoplasm and is a component of 40S ribosomal subunits in mammalian, Arabidopsis, and Urechis caupo cells (Auth and Brawerman, 1992;GarciaHernandez et al, 1994;Tohgo et al, 1994;Rosenthal and Wordeman, 1995). It has also been proposed that p40 is detected in developing mice retina and participates in defining the dorsal/ventral axis in developing retinas (Rabacchi et al, 1990;McCaffery et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

The induction of apoptosis in HeLa cells by the loss of LBP-p40

Kaneda

Kinoshita

et al. 1998

Cell Death Differ

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To analyze the function of the laminin-binding protein precursor p40 (LBP-p40) in higher eukaryotic cells, plasmid DNA expressing antisense or sense cDNA for p40 under the control of the LacSwitch system was introduced into HeLa cells. Stable transformants were isolated, and the expression of p40 was assayed by Western and Northern blotting. The expression level of p40 was not affected in HeLa cell transformants cultured in 10% serum-supplemented media with the induction of antisense (AS)-p40 with 5 mM IPTG. However, both the protein and message for endogenous p40 in serum-depleted media with 5 mM IPTG were reduced to about 30 ± 10% of the expression level in serum-free media without 5 mM IPTG. Colony formation was inhibited with the suppression of p40. AS-p40 clones died in 7 days when cultured in serum-depleted media with 5 mM IPTG, while clones without 5 mM IPTG AS-p40 clones never died, even in serum-depleted media. Additionally, sense (S)-p40 clones and control CAT clones survived more than 2 weeks in serum-free media with 5 mM IPTG. DNA fragmentation assay revealed that cell death induced by the reduction of AS-p40 resulted from apoptosis. Both the inhibition of cell growth and apoptotic cell death were partially rescued by the transfer of the p40 cDNA expression vector to AS-p40 clones. Moreover, the introduction of a synthetic hammerhead ribozyme for LBP-p40 using a fusigenic viral liposome suppressed the message for LBP-p40 even in the presence of 10% serum, and it also induced apoptosis.

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“…Identification of FSBA-labeled Peptide-0.2 mg of CD38 was labeled with 1 mM FSBA for 4 h and subjected to proteolysis by lysylendopeptidase (Wako Pure Chemical Industries, Osaka, Japan) as described previously (31). The lysylendopeptidase fragments were separated by reverse-phase HPLC using a RP C2/C18 column (3.2 ϫ 30 mm, Pharmacia Biotech) at a flow rate of 200 l/min.…”

Section: Methodsmentioning

confidence: 99%

“…An aliquot (10 l) of each peak was blotted on a polyvinylidene difluoride membrane and analyzed for the presence of FSBA-labeled peptide by Western blot using an anti-FSBA antibody as described above. The peak containing the FSBAlabeled peptide was subjected to automated Edman degradation as described previously (31).…”

Section: Methodsmentioning

confidence: 99%

Lysine 129 of CD38 (ADP-ribosyl Cyclase/Cyclic ADP-ribose Hydrolase) Participates in the Binding of ATP to Inhibit the Cyclic ADP-ribose Hydrolase

Tohgo¹,

Munakata

Takasawa

et al. 1997

Journal of Biological Chemistry

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CD38 catalyzes not only the formation of cyclic ADPribose (cADPR) from NAD؉ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD ؉ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.Cyclic ADP-ribose (cADPR) 1 (1) induces the release of Ca 2ϩ from microsomes in a variety of tissues and cells including pancreatic ␤ cells (2-10). cADPR is synthesized from NAD ϩ by ADP-ribosyl cyclase, which was purified as a soluble 29-kDa protein from Aplysia ovotestes (11-13). The amino acid sequences of Aplysia ADP-ribosyl cyclases (14, 15) showed a high degree of homology with that of CD38 (16, 17), which was reported to be a surface antigen of human lymphocytes (18). From the experiments in which CD38 cDNA was expressed in mammalian cells, CD38 was shown to catalyze not only the formation of cADPR from NAD ϩ but also the hydrolysis of cADPR to ADP-ribose (ADPR) (19 -21). We have demonstrated that ATP inhibited the hydrolysis, resulting in the accumulation of cADPR (19). Furthermore, we produced transgenic mice overexpressing human CD38 in pancreatic ␤ cells and demonstrated that ATP, produced in the process of glucose metabolism, increased the accumulation of cADPR to enhance the Ca 2ϩ mobilization from the intracellular stores for insulin secretion in the transgenic islets (22).In the present study, we expressed human CD38 in Escherichia coli and purified it to homogeneity. Using the purified CD38, we found that Lys-129 of CD38 participated in cADPR binding and that ATP competed with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38. EXPERIMENTAL PROCEDURESPurification of Soluble CD38 -We isolated a CD38 cDNA from an insulinoma of a Japanese patient by reverse transcriptase-polymerase chain reaction (PCR) (19) and used it for functional analyses of CD38 (17,19,22). The cDNA sequence was exactly the same as the CD38 sequence reported by Jackson and Bell (18) except for two base substitutions (nucleotide 213, A 3 C, and nucleotide 215, C 3 A) (19). The substitutions were also found in the corresponding...

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Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit

Cited by 38 publications

References 23 publications

siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis

siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis

The induction of apoptosis in HeLa cells by the loss of LBP-p40

Lysine 129 of CD38 (ADP-ribosyl Cyclase/Cyclic ADP-ribose Hydrolase) Participates in the Binding of ATP to Inhibit the Cyclic ADP-ribose Hydrolase

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