CDC2L5, a Cdk-like kinase with RS domain, interacts with the ASF/SF2-associated protein p32 and affects splicing in vivo.

The human CDC2L5 gene encodes a protein of unknown physiological function. This protein is closely related to the cyclin-dependent kinase (Cdks) family and contains an arginine/serine-rich (RS) domain. The Cdks were first identified as crucial regulators of cell-cycle progression, more recently they were found to be involved in transcription and mRNA processing. RS domains are mainly present in proteins regulating pre-mRNA splicing, suggesting CDC2L5 having a possible role in this process. In this study, we demonstrate that CDC2L5 is located in the nucleoplasm, at a higher concentration in speckles, the storage sites for splicing factors. Furthermore, this localization is dependent on the presence of the N-terminal sequence including the RS domain. Then, we report that CDC2L5 directly interacts with the ASF/SF2-associated protein p32, a protein involved in splicing regulation. Overexpression of CDC2L5 constructs disturbs constitutive splicing and switches alternative splice site selection in vivo. These results argue in favor of a functional role of the CDC2L5 kinase in splicing regulation.

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.