Joas-Sander, Suzane; (2000) In vitro function and interactions of the actin-associated protein caplin: A member of transgelin gene family. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Caplin is a member of the Transgelin (Tg) multigene family of actin binding proteins (ABPs) associated with the actin filament network that regulates the physical status and interactions of actin. Caplin is the second member of a polypeptide doublet of 22KDa originally identified on a SDS-PAGE by a monoclonal antibody and named PC4L (Shapland, 1988). The upper isoform of this doublet, Transgelin (Tg), was purified from sheep aorta and was found to cross-link actin filaments in vitro (Shapland et al., 1993). Transgelin was found to be down regulated by oncogenic transformation and changes in cell shape (Shapland et al., 1993). Caplin is present in all cells thus far examined, including transformed fibroblasts and lymphocytes, and in these cells, Caplin was found to be the only member of the PC4 doublet present The cDNA sequence of Caplin was obtained from human T cell lymphoma (HTCL) and was found to localize, by light microscopy, along actin filaments (Martin Smith PhD thesis). The in vitro function of this isoform was unknown and the objectives of my thesis was to purify and investigate the in vitro function of protein Caplin in normal cells. To allow this objective, I generated a pGEX fusion protein. Caplin cDNA sequence was obtained by RTPCR of mRNA obtained from normal mouse thymocytes using oligonucleotides derived from HTCL cDNA sequence (Martin Smith, PhD Thesis). Fusion protein was obtained by in frame ligation of Caplin cDNA to an EcoRI site in the bacterial expression vector pGEX-4T-3. Purification of fusion protein was achieved by chromatography followed by thrombin cleavage of fusion protein Caplin. The translated product of normal mouse thymus Caplin open reading frame consist of 199 amino acids with a calculated molecular weight of 22.391Da, an estimated PI of 8.41 and an extinction coefficient of 1.302. Caplin cDNA sequence analysis indicated the presence of a putative nuclear localisation signal, two potential EF-hand sites (23-34 and 108-118) and protein kinase C phosphorylation (180-182) site. Database searches indicates that Caplin amino acid sequence is highly homologous to protein NP25 (83%), Transgelin (82%) and Calponin (58%) and significantly homologous to sm20 (56%), VAV (54%) and UNC87 (48%). Further analysis indicates that Caplin has the same modular structure as found in the calponin family, consisting of an amino terminal CH domain (1-134), followed by an ABS domain (152-173) and a carboxyl terminal R domain (174-198). Cell permeabilization and immunofluorescence showed that Caplin binds directly to actin filaments and also indicated staining at nuclear level. In vitro functional studies including low shear viscometry, fluorimetry, sucrose density and sedimentation assays indicates that Caplin is a monomeric protein that binds to actin filaments with an estimated binding constant of Ka=4×10^5M-1, and acts in vitro as a calcium independent actin barbed end capping protein. Because this protein caps the barbed ends of actin filaments and is a member of the Transgelin family of actin binding proteins, I named it Caplin.

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