RESUMO
Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Jejuno/metabolismo , Miócitos de Músculo Liso/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Adenoviridae/genética , Motivos de Aminoácidos , Animais , Membrana Celular/química , Movimento Celular , Regulação da Expressão Gênica , Vetores Genéticos , Jejuno/citologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Quinase de Cadeia Leve de Miosina/química , Quinase de Cadeia Leve de Miosina/genética , Fosforilação , Cultura Primária de Células , Ligação Proteica , Transdução de Sinais , Tensão Superficial , TransfecçãoRESUMO
Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K(+)-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.