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1.
J Biol Chem ; 288(49): 35604-16, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24165133

RESUMO

Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Adesões Focais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Células Cultivadas , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Transferência Ressonante de Energia de Fluorescência , Técnicas de Inativação de Genes , Humanos , Queratinócitos/metabolismo , Camundongos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína
2.
Methods Enzymol ; 426: 69-84, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17697880

RESUMO

Integrin adhesion receptors mediate cell-cell and cell-substratum adhesion and provide a continuous link for the bidirectional transmission of mechanical force and biochemical signals across the plasma membrane. Integrin-dependent cellular activities such as adhesion, migration, proliferation, and survival rely upon the dynamic interaction of integrin cytoplasmic tails with intracellular integrin-binding proteins. In this review, we describe some of the methods that we have used to identify and characterize the interactions between integrin cytoplasmic tails and cytoskeletal proteins, as well as highlight methods to decipher the regulation of integrin tail interactions with intracellular ligands. Specifically, we describe recombinant models of integrin cytoplasmic tails and their use in protein-protein interaction studies.


Assuntos
Citoesqueleto/metabolismo , Integrinas/metabolismo , Animais , Humanos
3.
Cancer Genet Cytogenet ; 175(1): 61-4, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17498560

RESUMO

Loss or mutation of known tumor suppressor genes accounts for a small proportion of all breast cancers. We have recently shown that interferon regulatory factor 1 (IRF1) is a putative tumor suppressor gene in breast cancer. We now report that the A4396G single nucleotide polymorphism in the IRF1 gene is more frequent in human breast cancer cell lines than in the general population (P = 0.01). Furthermore, A4396G is more frequently expressed in African American (black) than in European ancestry (white) subjects (n = 70 subjects; P < or = 0.001), leading to a significant difference in genotype distribution between these populations (P = 0.002).


Assuntos
Fator Regulador 1 de Interferon/genética , Polimorfismo de Nucleotídeo Único , Negro ou Afro-Americano/genética , Alelos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Células HeLa , Humanos , Células Jurkat , Células K562 , Polimorfismo de Fragmento de Restrição , População Branca/genética
4.
Methods Mol Biol ; 757: 325-47, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21909921

RESUMO

Integrin adhesion receptors are essential for the development and functioning of multicellular animals. Integrins mediate cell adhesion to the extracellular matrix and to counter-receptors on adjacent cells, and the ability of integrins to bind extracellular ligands is regulated in response to intracellular signals that act on the short cytoplasmic tails of integrin subunits. Integrin activation, the rapid conversion of integrin receptors from low to high affinity, requires binding of talin to integrin ß tails and, once bound, talin provides a connection from activated integrins to the actin cytoskeleton. A wide range of experimental approaches have contributed to the current understanding of the importance of talin in integrin signaling. Here, we describe two methods that have been central to our investigations of talin; a biochemical assay that has allowed characterization of interactions between integrin cytoplasmic tails and talin, and a fluorescent-activated cell-sorting procedure to assess integrin activation in cultured cells expressing talin domains, mutants, dominant negative constructs, or shRNA.


Assuntos
Integrinas/metabolismo , Transdução de Sinais , Talina/metabolismo , Animais , Bioensaio , Células CHO , Cricetinae , Citometria de Fluxo , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
5.
J Biol Chem ; 284(17): 11485-97, 2009 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-19240021

RESUMO

Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Talin binding to integrin beta tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin beta tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with beta1 and beta3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin beta tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr(795)), a preceding threonine-containing region (Thr(788) and Thr(789)) of the integrin beta1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp(612) and kindlin-2 Trp(615)) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin beta3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous alpha5beta1 or stably expressed alphaIIbbeta3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate alphaIIbbeta3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate beta1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.


Assuntos
Cadeias beta de Integrinas/química , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Estrutura Terciária de Proteína
6.
J Mol Biol ; 394(5): 944-56, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19804783

RESUMO

The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the beta-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced alphaIIbbeta3 integrin activation and for the localization of kindlin-1 to focal adhesions.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sequência de Aminoácidos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas de Neoplasias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Talina/química , Talina/genética , Talina/metabolismo
7.
J Biol Chem ; 283(50): 35154-63, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18829455

RESUMO

A link between sites of cell adhesion and the cytoskeleton is essential for regulation of cell shape, motility, and signaling. Migfilin is a recently identified adaptor protein that localizes at cell-cell and cell-extracellular matrix adhesion sites, where it is thought to provide a link to the cytoskeleton by interacting with the actin cross-linking protein filamin. Here we have used x-ray crystallography, NMR spectroscopy, and protein-protein interaction studies to investigate the molecular basis of migfilin binding to filamin. We report that the N-terminal portion of migfilin can bind all three human filamins (FLNa, -b, or -c) and that there are multiple migfilin-binding sites in FLNa. Human filamins are composed of an N-terminal actin-binding domain followed by 24 immunoglobulin-like (IgFLN) domains and we find that migfilin binds preferentially to IgFLNa21 and more weakly to IgFLNa19 and -22. The filamin-binding site in migfilin is localized between Pro(5) and Pro(19) and binds to the CD face of the IgFLNa21 beta-sandwich. This interaction is similar to the previously characterized beta 7 integrin-IgFLNa21 interaction and migfilin and integrin beta tails can compete with one another for binding to IgFLNa21. This suggests that competition between filamin ligands for common binding sites on IgFLN domains may provide a general means of modulating filamin interactions and signaling. In this specific case, displacement of integrin tails from filamin by migfilin may provide a mechanism for switching between different integrin-cytoskeleton linkages.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas Contráteis/química , Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto/metabolismo , Cadeias beta de Integrinas/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Animais , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Filaminas , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Conformação Molecular , Células NIH 3T3 , Ligação Proteica
8.
Carcinogenesis ; 26(9): 1527-35, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15878912

RESUMO

We have directly assessed the ability of interferon regulatory factor-1 (IRF-1) to act as a tumor suppressor gene in human breast cancer cells and explored whether this suppressor function is mechanistically conferred by affecting cell cycle transition, apoptosis and/or caspase activation. We have used a dual approach, measuring whether overexpression of wild-type IRF-1 or a dominant negative IRF-1 (dnIRF-1) produce opposing effects on breast cancer cell proliferation in vitro or tumorigenicity in athymic nude mice. Mechanistic studies determined the effects of blocking endogenous IRF-1 expression on cell cycle transition by flow cytometry, on apoptosis by Annexin V staining, and on caspase activation by fluorescent substrate cleavage. IRF-1 mRNA (P < or = 0.001) and protein (P < or = 0.001) are highly expressed in non-tumorigenic, normal, mammary epithelial cells, with intermediate expression in tumorigenic, but non-metastatic, cells and very low expression in metastatic cell lines. In MCF-7 cells transfected with a wild-type IRF-1 (MCF-7/IRF-1), IRF-1 mRNA expression inversely correlates with the rate of cell proliferation (r = -0.91; P = 0.002). Conversely, expression of dnIRF-1 in both MCF-7 (MCF-7/dnIRF-1; p53 wild-type) and T47D cells (T47D/dnIRF-1; p53 mutant) increases cell proliferation (P < or = 0.001). In athymic nude mice, the incidence of MCF-7/IRF-1 xenografts is reduced (P = 0.045), whereas MCF-7/dnIRF-1 xenografts exhibit a significantly higher tumor incidence (P < or = 0.001). Effects of IRF-1/dnIRF-1 are mediated through changes in the rates of apoptosis and not through cell cycle regulation. MCF-7/dnIRF-1 cells exhibit a 50% decrease in basal apoptosis (P = 0.007) and a significant reduction in caspase 8 activity (P = 0.03); similar effects occur in T47D/dnIRF-1 cells, where the effects on apoptosis appear to be mediated through inhibition of caspases 3/7 (P < 0.001) and caspase 8 (P = 0.03). These data establish a functional role for IRF-1 in the growth suppression of breast cancer cells and strongly implicate IRF-1 as a tumor suppressor gene in breast cancer that acts, independent of p53, to control apoptosis.


Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Fosfoproteínas/genética , Apoptose , Mama/citologia , Mama/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Caspases/metabolismo , Ciclo Celular , Células Epiteliais/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon , Cinética , RNA Mensageiro/genética , Supressão Genética
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