RESUMO
KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas do Citoesqueleto/química , Adesões Focais/química , Complexos Multiproteicos/química , Talina/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Actomiosina/química , Actomiosina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Matriz Extracelular/química , Matriz Extracelular/genética , Adesões Focais/genética , Células HeLa , Humanos , Integrinas/química , Integrinas/genética , Fenômenos Mecânicos , Mecanotransdução Celular/genética , Microtúbulos/química , Microtúbulos/genética , Complexos Multiproteicos/genética , Contração Muscular/genética , Resistência ao Cisalhamento/fisiologia , Talina/genéticaRESUMO
Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain-containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif-containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Chaperonas Moleculares/metabolismo , Pseudópodes/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular , Clonagem Molecular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Talina/genéticaRESUMO
Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the "LC8Pred" algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with â¼78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome.
Assuntos
Dineínas do Citoplasma/metabolismo , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Algoritmos , Calorimetria , Proteínas de Ciclo Celular/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Ligação Proteica , Proteômica , TermodinâmicaRESUMO
The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.
RESUMO
Microtubules regulate signaling, trafficking, and cell mechanics, but the respective contribution of these functions to cell morphogenesis and migration in 3D matrices is unclear. Here, we report that the microtubule plus-end tracking protein (+TIP) SLAIN2, which suppresses catastrophes, is not required for 2D cell migration but is essential for mesenchymal cell invasion in 3D culture and in a mouse cancer model. We show that SLAIN2 inactivation does not affect Rho GTPase activity, trafficking, and focal adhesion formation. However, SLAIN2-dependent catastrophe inhibition determines microtubule resistance to compression and pseudopod elongation. Another +TIP, CLASP1, is also needed to form invasive pseudopods because it prevents catastrophes specifically at their tips. When microtubule growth persistence is reduced, inhibition of depolymerization is sufficient for pseudopod maintenance but not remodeling. We propose that catastrophe inhibition by SLAIN2 and CLASP1 supports mesenchymal cell shape in soft 3D matrices by enabling microtubules to perform a load-bearing function.
Assuntos
Mesoderma/metabolismo , Mesoderma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colágeno/metabolismo , Exocitose , Feminino , Adesões Focais/metabolismo , Células HEK293 , Humanos , Interfase , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Polimerização , Pseudópodes/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5ß and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.