RESUMEN
The Cdo-p38MAPK (p38 mitogen-activated protein kinase) signaling pathway plays important roles in regulating skeletal myogenesis. During myogenic differentiation, the cell surface receptor Cdo bridges scaffold proteins BNIP-2 and JLP and activates p38MAPK, but the spatial-temporal regulation of this process is largely unknown. We here report that KIF5B, the heavy chain of kinesin-1 motor, is a novel interacting partner of BNIP-2. Coimmunoprecipitation and far-Western study revealed that BNIP-2 directly interacted with the motor and tail domains of KIF5B via its BCH domain. By using a range of organelle markers and live microscopy, we determined the endosomal localization of BNIP-2 and revealed the microtubule-dependent anterograde transport of BNIP-2 in C2C12 cells. The anterograde transport of BNIP-2 was disrupted by a dominant-negative mutant of KIF5B. In addition, knockdown of KIF5B causes aberrant aggregation of BNIP-2, confirming that KIF5B is critical for the anterograde transport of BNIP-2 in cells. Gain- and loss-of-function experiments further showed that KIF5B modulates p38MAPK activity and in turn promotes myogenic differentiation. Of importance, the KIF5B-dependent anterograde transport of BNIP-2 is critical for its promyogenic effects. Our data reveal a novel role of KIF5B in the spatial regulation of Cdo-BNIP-2-p38MAPK signaling and disclose a previously unappreciated linkage between the intracellular transporting system and myogenesis regulation.
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular , Cinesinas/metabolismo , Sistema de Señalización de MAP Quinasas , Mioblastos/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Proteínas Portadoras/genética , Línea Celular , Endosomas/metabolismo , Humanos , Cinesinas/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Desarrollo de Músculos , Unión Proteica , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Mechanotransduction is a critical function for cells, in terms of cell viability, shaping of tissues, and cellular behavior. In vitro, cellular level forces can stretch adhesion proteins that link extracellular matrix to the actin cytoskeleton exposing hidden binding sites. However, there is no evidence that in vivo forces produce significant in vivo stretching to cause domain unfolding. We now report that the adhesion protein, talin, is repeatedly stretched by 100-350 nm in vivo by myosin contraction of actin filaments. Using a functional EGFP-N-Talin1-C-mCherry to measure the length of single talin molecules, we observed that the C-terminal mCherry was normally displaced in the direction of actin flow by 90 to >250 nm from N-EGFP but only by 50-60 nm (talin's length in vitro) after myosin inhibition. Individual talin molecules transiently stretched and relaxed. Peripheral, multimolecular adhesions had green outside and red proximal edges. They also exhibited transient, myosin-dependent stretching of 50-350 nm for 6-16 s; however, expression of the talin-binding head of vinculin increased stretching to about 400 nm and suppressed dynamics. We suggest that rearward moving actin filaments bind, stretch, and release talin in multiple, stochastic stick-slip cycles and that multiple vinculin binding and release cycles integrate pulling on matrices into biochemical signals.
Asunto(s)
Mecanotransducción Celular , Talina/biosíntesis , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Adhesión Celular , Línea Celular , Chlorocebus aethiops , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Talina/metabolismoRESUMEN
The BNIP-2 and Cdc42GAP homology (BCH) domain is a novel regulator for Rho GTPases, but its impact on p50-Rho GTPase-activating protein (p50RhoGAP or Cdc42GAP) in cells remains elusive. Here we show that deletion of the BCH domain from p50RhoGAP enhanced its GAP activity and caused drastic cell rounding. Introducing constitutively active RhoA or inactivating GAP domain blocked such effect, whereas replacing the BCH domain with endosome-targeting SNX3 excluded requirement of endosomal localization in regulating the GAP activity. Substitution with homologous BCH domain from Schizosaccharomyces pombe, which does not bind mammalian RhoA, also led to complete loss of suppression. Interestingly, the p50RhoGAP BCH domain only targeted RhoA, but not Cdc42 or Rac1, and it was unable to distinguish between GDP and the GTP-bound form of RhoA. Further mutagenesis revealed a RhoA-binding motif (residues 85-120), which when deleted, significantly reduced BCH inhibition on GAP-mediated cell rounding, whereas its full suppression also required an intramolecular interaction motif (residues 169-197). Therefore, BCH domain serves as a local modulator in cis to sequester RhoA from inactivation by the adjacent GAP domain, adding to a new paradigm for regulating p50RhoGAP signaling.
Asunto(s)
Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/metabolismo , Estructura Terciaria de Proteína , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Activadoras de GTPasa/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation.