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Vinculin is a 117 kDa protein localized in the cytoplasm. It is a component of integrin- mediated cell-matrix adhesion that includes assembly, strength and turnover of focal adhesion. Vinculin also localizes in catherin-mediated cell-cell junctions as a functional regulator (Carisey and Ballestrem, 2011; Peng et al., 2011). Now, vinculin was also known to act as a regulator of cellular machineries for the cell growth, cell shape change and in phagocytosis (Borgon et al., 2004). The function of vinculin is highly regulated by the conformational changes in its structure but it is devoid of enzymatic activity (Peng et al., 2011). Loss of vinculin in mouse embryonic fibroblasts in vinculin knock-out mice and also from vinculin null F9 embryonic carcinoma cells were fragile and show increased cell motility than its wild-type counter parts. Thus, it was speculated that loss of vinculin result in metastases of tumors (Carisey and Ballestrem, 2011). The electron microscope (EM) studies shows that vinculin is a globular protein having a 90 kDa N-terminal globular head, a short proline-rich linker and a 27 kDa C-terminal rod shaped tail. All these regions of vinculin have interaction sites for numerous binding partners (Peng et al., 2011; Ziegler et al., 2006). Vinculin in its activated state interacts with 19 different binding factors like talin, α-actin, α/β-catenin, α/β-vinexin, CAP (c-Cb1-Associated Protein), nArgBP2, VASP (Vasodilator-Stimulated Phosphoprotein), Arp2/3, Paxillin, Hic-5, F-actin, Protein kinase Cα (PKCα), synemin, calpain, polycystin-1, raver1 and PIP2 (Phosphatidylinositol-4,5-bisphosphate) (Carisey and Ballestrem, 2011).
The crystal structure of full length chicken vinculin was solved in 3.1AÌŠ resolution (Bakolitsa et al., 2004) and the crystal structure of full length human vinculin was solved in 2.85 AÌŠ resolutions (Borgon et al., 2004). Both the crystal structure studies reveal that the vinculin contains eight anti-parallel α-helical bundles that are organized to five domain structure namely domain D1-D5 in case of chicken vinculin (Bakolitsa et al., 2004) and Vh 1-3, Vt 1-2 in case of human vinculin(Borgon et al., 2004). The vinculin head comprises of D1-D3, D4 is the proline-rich linker neck that is linked to the tail domain D5 or Vt (Bakolitsa et al., 2004; Ziegler et al., 2006). The α-helical bundles of vinculin protrude out of the molecule for the binding of its partners (Borgon et al., 2004). Among all the binding partners, few proteins that include talin, α-catenin, β-catenin, α-actinin and IPaA binds to the head domain of vinculin. The proteins VASP, Arp2/3, vinexin and ponsin binds to the ligands present in proline rich linker. The tail domain of vinculin interacts with paxillin, F-actin, PIP2, PKCα and raver1. Vinculin exist in two conformations i. e. auto-inhibited or inactive conformation and an open or active conformation. The binding sites of the ligands are blocked in the inactive conformation while the binding sites for the partners are exposed in the active conformation. The change in the conformation of vinculin is studied by FRET (Forster resonance energy transfer). The FRET probe is a N-terminus CFP tagged vinculin construct along with YFP inserted immediately after D5. In the inactive conformation, these two fluorophores produce high FRET signal due to their close proximity but the intensity of FRET signal gets reduced in the active state of vinculin due to the separation of fluorophores (Peng et al., 2011).
Although vinculin is well known as a cytoplasmic protein but there are few reports on the nuclear localization of vinculin. Marquez and his colleagues observed the presence of vinculin in both the cytoplasmic membrane and nuclear fractions during the investigation of cellular distribution of vinculin in the primary cultured collecting duct cells. They have also observed a high vinculin signal in the nuclear preparations (Marquez et al., 2008).
Here, in this study, we have also report the presence of vinculin in the nuclear fractions of chicken liver and it is purified to the homogeneity. The Maldi-TOF/TOF analysis revealed the purified protein is vinculin.
There are ample reports on characterization of cytoplasmic vinculin from a number of organisms. However, there are very few reports depicting nuclear localization of vinculin.
Purification of vinculin
Since vinculin do not demonstrate any activity based assay, western blotting was employed to track vinculin during different steps of purification. The chicken liver tissue was homogenized and fractionated into the nuclear and cytoplasmic fractions. Subsequently, cytoplasmic and nuclear extracts were prepared by standard methods. Equal amount in terms of protein of the cytoplasmic and nuclear extracts were separated on SDS PAGE, transferred to PVDF membrane and were probed with anti-vinculin antibody. It was seen that an immunoreactive band corresponding to vinculin was observed both in the nuclear as well as the cytoplasmic extracts (Fig. 1a). Densitomatric comparison of the band intensities revealed that approximately . Anti GAPDH antibody acted as loading controls. The we