Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells.
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, alpha-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 [(35)S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, beta subunit (Cct2); heterogeneous 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.
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
Kazuhiro
K
Nishimura
Hiroyuki
H
Okudaira
Eriko
E
Ochiai
Kyohei
K
Higashi
Mayumi
M
Kaneko
Itsuko
I
Ishii
Tomoe
T
Nishimura
Naoshi
N
Dohmae
Keiko
K
Kashiwagi
Kazuei
K
Igarashi
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002474:Cell-Free System; D000518:Eflornithine / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000737:chemistry; D009690:Nucleic Acid Conformation; D011073:Polyamines / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D020121:5' Untranslated Regions; D009363:Neoplasm Proteins; D011073:Polyamines; D011993:Recombinant Fusion Proteins; D000518:Eflornithine