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 l ength rCdc5 sequence (2847 bp) was obtained by 5'-RACE and cytokine regulat ion of Cdc5 expression was examined. PRL and interleukin-2 (IL2) act as mit ogens in Nb2 T-lymphoma cells. Fibroblast growth factor (FGF-2) is not mito genic 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 rem ained 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 cel ls. PRL or FGF-2 treatment transiently increased nuclear Cdc5-specific immu nofluorescence at 4 It but IL-2 gave maximal nuclear accumulation of Cdc5 a t 24 h. The deduced rCdc5 protein has similar to 98% amino acid identity wi th human Cdc5. Like other Cdc5 family members. the N-terminus of rCdc5 cont ains two repeats of a DNA-binding domain found in a-, b- and c-Myb. Gel shi ft assays using P-32-labeled Myb consensus oligonucleotides revealed two My b-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 partial ly inhibited by anti-Myb or anti-Cdc5 antibodies. In summary, rapid activat ion of Cdc5 in response to mitogenic and non-mitogenic stimuli suggests a c omplex role for Cdc5 in cellular regulation and this may not be restricted to mitotic entry or G2/M progression as previously supposed. (C) 2001 Elsev ier Science Ireland Ltd. All rights reserved.