Prolactin, interleukin-2 and FGF-2 stimulate expression, nuclear distribution and DNA-binding of rat homolog of pombe Cdc5 in Nb2 T lymphoma cells.
Pombe and human Cdc5 have been implicated in G2/M progression, but recently Cdc5 was identified as a component of a multiprotein complex essential for pre-mRNA splicing. We have previously isolated a prolactin (PRL)-inducible partial cDNA (1907 bp) encoding rat Cdc5. In the present study, the full length rCdc5 sequence (2847 bp) was obtained by 5'-RACE and cytokine regulation of Cdc5 expression was examined. PRL and interleukin-2 (IL2) act as mitogens in Nb2 T-lymphoma cells. Fibroblast growth factor (FGF-2) is not mitogenic in Nb2 cells but inhibits apoptosis of PRL-deprived cells. This study showed that PRL, IL-2 and FGF-2 rapidly increased Nb2 Cdc5 expression (3.4 kb mRNA) to reach 2-3-fold above controls at 4 h, and Cdc5 mRNA levels remained elevated at 24 h. There was a corresponding 2-3-fold increase in Cdc5 protein (105 kDa) levels at 24 h. Immunoblotting and fluorescent confocal microscopy showed predominant nuclear/perinuclear Cdc5 in quiescent Nb2 cells. PRL or FGF-2 treatment transiently increased nuclear Cdc5-specific immunofluorescence at 4 h but IL-2 gave maximal nuclear accumulation of Cdc5 at 24 h. The deduced rCdc5 protein has approximately 98% amino acid identity with human Cdc5. Like other Cdc5 family members, the N-terminus of rCdc5 contains two repeats of a DNA-binding domain found in a-, b- and c-Myb. Gel shift assays using (32)P-labeled Myb consensus oligonucleotides revealed two Myb-specific DNA-protein complexes in Nb2 nuclear extracts. Formation of both complexes was increased by PRL or FGF-2 at 1-5 and at 20 h and was partially inhibited by anti-Myb or anti-Cdc5 antibodies. In summary, rapid activation of Cdc5 in response to mitogenic and non-mitogenic stimuli suggests a complex role for Cdc5 in cellular regulation and this may not be restricted to mitotic entry or G2/M progression as previously supposed.
Johnson
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LM
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, NS B3H 4H7, Canada.
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D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D007376:Interleukin-2 / Q000494:pharmacology; D016399:Lymphoma, T-Cell / Q000473:pathology; D008969:Molecular Sequence Data; D011388:Prolactin / Q000494:pharmacology; D051381:Rats / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D007376:Interleukin-2; D029702:Schizosaccharomyces pombe Proteins; C085875:cdc5 protein, S pombe; D016222:Fibroblast Growth Factor 2; D011388:Prolactin; D004247:DNA