Studies On The Interaction Between Kindlin-2and Prdx4

Kindlin protein family is a family of focal adhesion proteins firstly discovered in early1950s. Till now, three subfamily members, i.e., Kindlin-1, Kindlin-2and Kindlin-3, have been identified. They have been confirmed to be associated with dermatological diseases, vasculogenesis, immunoresponse, tumor invasion and drug resistance of cancer cells. Studies on Kindlins have shown that by interacting with integrins, Kindlins play an important role in cell adhesion. Serving as signaling molecules, Kindlins participate in the regulation of cell adhesion, migration, proliferation and differentiation.Kindlin family members not only share a high degree of homology, but also are highly conservative. Kindlin-2shares62%amino acid identity with Kindlin-1. Kindlin-3shares49%amino acid identity with Kindlin-1, and Kindlin-2shares53%amino acid identity with Kindlin-3. The Kindlin proteins are composed of constant regions and variation areas. Kindlin proteins have a unique domain architecture in the constant region. They are composed of a centrally located FERM domain interrupted by a PH domain. FERM domains are found in a number of proteins linking the membrane to the cytoskeleton. Among these proteins, the FERM domain of talin is most homologous to the Kindlin FERM domains. It has been proposed that Kindlins bind to β1and P3integrins with a similar mechanism as it has been described for talin domain interrupted by a pleckstrin homology (PH) domain. This construction is sometimes seen in skeleton proteins. And the construction is composed of three domains which are Fl1F2and F3, the FO domain is prior to the structure of the core domain. The F3domain of Kindlins is similar to F3in the FERM domain of talin. Phosphotyrosine-binding domain (PTB) can also be combined with (33cytoplasmic tail region of integrin.Kindlin-2appeared to play an important role in focal adhesions. The N-terminal region of migfilin binds to filamin, and could provide a means for Kindlin-2to connect to the actin cytoskeleton via recruiting migfilin/filamin complexes. Depletion of Kindlin-2blocks recruitment of migfilin to focal adhesions, whereas the localization of paxillin, a well-known focal adhesion marker, is not affected.β2integrins carry a phenylalanine at the position of the distal tyrosine of β1/β3cytoplasmic tails which still permits Kindlin-3binding. A large number of proteins can interact with (3integrin cytoplasmic tails; however, before the discovery of Kindlins, only talin was demonstrated unequivocally to trigger integrin activation, i.e. an increase in integrin binding affinity, with the evidence of overexpression of talin seemed to be sufficient for integrin activation. Kindlin is necessary to activate integrins came from Kindlin-3knockout studies in mice.Kindlin-2may play an important role in the tumorigenesis and progression of cancers. Many studies have shown that the expression level of Kindlin-2is highly related to certain tumors. Ours earlier study has elucidated the expression level of Kindlin-2not only closely relates to the malignancy of prostate cancer, but also controls the sensitivity of prostate cancer cells to cisplatin. Moreover, Kindlin-2has different expression levels in lung cancer cell lines, and its high expression can significantly promote the migration of U-1752cells. Kindlins probably regulates the tumor invasion and metastasis process by its interaction with integrin.In our earlier effort on exploring new candidates of Kindlin-2interacting proteins, a pull-down assay was conducted by anti-Kindlin-2antibody to PC-3(human prostate carcinoma) cell lysates. With the analyses of2-D DIGE and MALDI TOF/TOF mass spectrometry, Prdx4a family member of peroxiredoxin, was found to be a potentially interesting interacting protein of Kindlin-2.Organisms living under aerobic conditions have developed various anti-oxidative mechanisms to protect them from damages by reactive oxygen species (ROS). A novel family of anti-oxidative proteins, designated as peroxiredoxin (Prdx), has been identified in the past two decades and currently comprises six members in mammals. They share a common reactive Cys residue in the N-terminal region, and are capable of serving as a peroxidase and involve thioredoxin and/or glutathione as the electron donor. Prdxl to Prdx4have an additional Cys residue in the conserved C-terminal region, and are cross members as judged by the amino acid sequence similarity. Prx5also contains an additional Cys in its C-terminal region which is less conserved. On the other hand, Prdx6has only one unique Cys. These Prdx family members are distributed in the cytosol, mitochondria, peroxisome and plasma, all of which are potential sites of ROS production. In addition to their role as a peroxidase, however, a body of evidence has accumulated to suggest that individual members also serve divergent functions which are associated with various biological processes such as the detoxification of oxidants, cell proliferation, differentiation and gene expression. It would be expected that these functions might not necessarily depend on peroxidase activity and, therefore, it seems likely that the divergence is due to unique molecular characteristics intrinsic to each member. A comparative study of the divergence would lead to a better understanding of the biological significance of the Prdx family. Peroxiredoxin (Prdx) protein family members are a high-abundance protein enzymes that contribute to in vivo H2O2level control and mediate cell signal transduction through regulating the redox state of protein kinases. Based on the cysteine residues conserved in the deduced amino acid sequence and their catalytic mechanisms, three subtypes of Prdx have been distinguished, namely,2-Cys Prdx, atycpical2-Cys Prx, and1-Cys Prdx. The activity of Prdx was regulated by polymerization, phosphorylation and proteolysis.Prdx1to Prdx4are cross members of the family and form ahomodimer with head-to-tail type association. The N-terminal Cys of the two conserved residues constitutes a catalytic center and is converted to Cys sulfenic acid (-SOH) via reaction with peroxides. The sulfenic acid is a reaction intermediate and immediately reacts with the C-terminal Cys in the other subunit, forming an intermolecular disulfide and water. Under inflammation caused by microbes, large amount of reactive oxygen species (ROS) are produced by activated inflammatory cells. However, normal body cells will be hurt if these oxidative products are over-induced or not eliminated efficiently. Thus, Prdx maintains dual character in its function. On one hand, acting as peroxiredoxin, Prdx holds a positive effect of clearing up ROS and thus protect normal cells from inflammatory harms. On the other hand, constantly exposure to environmental stress will damage both DNA and mitochondria. These damages probably reduce the capacity of DNA self-correction in cells and eventually lead to genetic mutation. Induced over-expression of Prdx4at this point could protect the cells from apoptosis and thus contribute to tumorigenesis. More and more evidences suggest Prdx expression level is closely related to tumor, and it has even been proposed that the Prdxs can be used as tumor markers.Therefore, we presume the interaction between Kindlin-2and Prdx4might be involved in the regulation of inflammation/stress-related tumorigenesis. In the present study, we have performed some experiments and obtained the following conclusions:1We have successfully constructed pEGFP-C3-Kindlin-2and pmcherry-N1-Prdx4fusion protein expression vectors.Human Prdx4and Kindlin-2full-length cDNAs were introduced to red fluorescent protein pmcherry and enhanced green fluorescent protein (EGFP) fusion protein expressing vectors, respectively. The resultant pmcherry-N1-Prdx4and pEGFP-C3-Kindlin-2constructs were then confirmed with different methods. These constructs can be used not only for localization studies, but also for functional studies and time-lapse experiments in the future.2We have validated the intracellular interaction between Kindlin-2and Prdx4.COS-7cells were transfected with FLAG-tagged Kindlin-2and HA-tagged Prdx4, and co-immunoprecipitation experiment showed H2O2treatment signicantly increased to stimulate theirthe interaction between Kindlin-2and Prdx4significantly increased before the stimulus. Endogenous co-immunoprecipitation experiments with arsenite-stimulated PC-3cells showed similar results, suggesting Kindlin-2and Prdx4can interact to each other in cells upon stresses.3Confocal microscopy studies indicated stress-induced co-localization of Kindlin-2and Prdx4.It was shown that pEGFP-C3-Kindlin-2distributed both in the cytoplasm and nucleus, and pmcherry-Nl-Prdx4was mainly in the cytoplasm. In resting cells, pEGFP-C3-Kindlin-2and pmcherry-Nl-Prdx4showed co-localization, which was enhanced significantly with NaAsO2stimulation. Endogenous co-localization experiments showed similar results.Through the above research, we come to the following conclusions:1pEGFP-C3-Kindlin-2and pmcherry-Nl-Prdx4fusion vectors were successfully constructed.2Exogenous co-immunoprecipitation experiments confirmed the interaction between Kindlin-2and Prdx4, which could be strongly enhanced by H2O2stimulation.3Co-immunoprecipitation experiments further confirmed arsenite-induced endogenousKindlin-2-Prdx4interaction.4Both exogenous and endogenous immunofluorescence experiments indicated arsenite-induced co-localization of Kindlin-2and Prdx4.