Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells |
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Authors: | Kazuhiro Nishimura Hiroyuki Okudaira Eriko Ochiai Kyohei Higashi Mayumi Kaneko Itsuko Ishii Tomoe Nishimura Naoshi Dohmae Keiko Kashiwagi Kazuei Igarashi |
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Affiliation: | aGraduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan;bAmine Pharma Research Institute, 1-8-15 Inohana, Chuo-ku, Chiba 260-0856, Japan;cBiomolecular Characterization Team, RIKEN Advanced Science Institute and CREST, JST, 2-1 Hirosawa, Wako 351-0198, Japan;dFaculty of Pharmacy, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan |
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Abstract: | In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, α-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [35S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, β subunit (Cct2); heterogenous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5′-UTR of Cct2 mRNA. |
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Keywords: | Polyamine modulon Ribosome shunting Cct2 Hnrpl Pgam1 |
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