Abstract
Cyclophilins of the Moca family (Cavarec, L., Kamphausen, T., Dubourg, B., Callebaut, I., Lemeunier, F., Metivier, D., Feunteun, J., Fischer, G., and Modjtahedi, N. (2002) J. Biol. Chem. 277, 41171-41182) are found only in organisms of the animal kingdom and share several structural and enzymatic features. The presence of serine/arginine (S/R) dipeptide repeats in their C-terminal tail suggests that these enzymes belong to the SR protein family involved in the regulation of gene expression. The function of this group of cyclophilins is currently unknown. However, their C-terminal tails contain a highly conserved polypeptide signature segment (the moca domain), which may well be involved in the functional regulation of these proteins. We report here the identification of five Cdc2-type phosphorylation sites gathered in and around the moca domain of SRcyp, a human cyclophilin belonging to the Moca family. The segment of SRcyp containing the identified sites is specifically phosphorylated in mitotic cells. This mitosis-specific phosphorylation was inhibited by treatment of the cells with roscovitine, a specific inhibitor of cyclin-dependent kinases, suggesting that the unknown activity of the moca domain of SRcyp requires mitotic regulation by the Cdc2-cyclin B kinase complex. The Cdc2-cyclin B complex was found to phosphorylate four of the five identified phosphorylation sites in vitro, providing further support for this possibility. Like many factors stored in nuclear speckles and involved in the regulation of gene expression, this nuclear cyclophilin displays a predominantly diffuse cytoplasmic distribution at the onset of mitosis. Only in late telophase is SRcyp recruited to the newly formed nuclei. The transit of SRcyp through mitotic interchromatin granule clusters, before re-entering the nucleus, suggests that the timing of the appearance of this cyclophilin in the telophasic nuclei is tightly coordinated with post-mitotic events. Human SRcyp is the first cell cycle-regulated cyclophilin to be described.
Highlights
We report here the cell cycle regulation of the nuclear cyclophilin SRcyp2 [14, 15], a human protein belonging to the Moca family
The Moca family member matrin-cyp has been shown to be a phosphoprotein [16], but our report is the first to address in detail the issue of specific phosphorylation events targeting this group of cyclophilins
We focused on the nuclear cyclophilin SRcyp [14, 15], a human protein belonging to the Moca family, and we demonstrated that the phosphorylation of this protein is regulated by the cell cycle
Summary
We obtained stably transfected clones of human U2OS cells producing a FLAG-tagged version of the CAD fragment (CAD-FLAG), synchronized them at various stages of the cell cycle (see “Experimental Procedures”), and prepared protein extracts, which we analyzed by Western blotting with an anti-FLAG mAb. The CAD-FLAG protein extracted from mitotic cells after nocodazole treatment clearly displayed an electrophoretic mobility shift with respect to the proteins extracted at other stages and from asynchronous growing cells (Fig. 2A, top panel). SRcyp Enters the Nuclei of Telophasic Cells after Nuclear Membrane Formation—As our results suggested that the activity of the nuclear cyclophilin SRcyp could be regulated by Cdc2-cyclin B, we investigated whether the cellular distribution of this protein changed during the course of mitosis.
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