RESUMO
A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Integrina alfa4/metabolismo , Animais , Western Blotting , Células CHO , Movimento Celular , Células Cultivadas , Cromatografia de Afinidade , Cricetinae , Cricetulus , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Cães , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Integrina alfa4/genética , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In vitro studies indicate that binding of talin to the beta(3) integrin cytoplasmic domain (tail) results in integrin alpha(IIb)beta(3) (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the beta(3) tail. We introduced a beta(3)(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a beta(3)(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate alpha(IIb)beta(3) require binding of talin to the beta(3) tail. beta(3)(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of beta(3)(Y747A) and virtually all beta(3)-null animals examined. Remarkably, less than 5% of beta(3)(L746A) animals exhibited this form of bleeding. These results establish that alpha(IIb)beta(3) activation in vivo is dependent on the interaction of talin with the beta(3) integrin cytoplasmic domain. Furthermore, they suggest that modulation of beta(3) integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.
Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Embolia Pulmonar/metabolismo , Talina/metabolismo , Trombose/metabolismo , Substituição de Aminoácidos , Anemia/genética , Anemia/metabolismo , Anemia/patologia , Animais , Plaquetas/patologia , Cloretos , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Compostos Férricos/toxicidade , Fibrinogênio/genética , Fibrinogênio/metabolismo , Filaminas , Hemorragia/genética , Hemorragia/metabolismo , Hemorragia/patologia , Homozigoto , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Mutação Puntual , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Embolia Pulmonar/induzido quimicamente , Embolia Pulmonar/genética , Embolia Pulmonar/patologia , Embolia Pulmonar/terapia , Talina/genética , Trombose/induzido quimicamente , Trombose/genética , Trombose/patologia , Trombose/terapiaRESUMO
Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly/disassembly. Talin, an actin and beta-integrin tail-binding protein, is essential for integrin activation and focal adhesion formation. Calpain-mediated cleavage of talin has a key role in focal adhesion turnover; however, the talin head domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions and maintains cell edge protrusions, suggesting that other steps, downstream of talin proteolysis, are required for focal adhesion disassembly. Here we show that talin head binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration, more tightly than full-length talin does and that this interaction leads to talin head ubiquitylation and degradation. We found that talin head is a substrate for Cdk5, a cyclin-dependent protein kinase that is essential for cell migration, synaptic transmission and cancer metastasis. Cdk5 phosphorylated talin head at Ser 425, inhibiting its binding to Smurf1, thus preventing talin head ubiquitylation and degradation. Expression of the mutant tal(S425A), which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitylation, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, talin head produced by calpain-induced cleavage of talin is degraded through Smurf1-mediated ubiquitylation; moreover, phosphorylation by Cdk5 regulates the binding of Smurf1 to talin head, controlling talin head turnover, adhesion stability and ultimately, cell migration.