RESUMEN
Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase 3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function, showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok-3/Grb2 complexes into BCR microsignalosomes and augmented phosphorylation of the inhibitory Lyn target SH2 domain-containing inositol 5' phosphatase. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Adaptadora GRB2/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Familia-src Quinasas/metabolismo , Aminoácidos/química , Animales , Calcio/metabolismo , Señalización del Calcio , Pollos , Activación Enzimática , Humanos , Activación de Linfocitos , Espectrometría de Masas/métodos , Microscopía Confocal/métodos , Transducción de SeñalRESUMEN
The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos B/metabolismo , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Calcio/metabolismo , Membrana Celular/genética , Activación Enzimática/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/genética , Quinasa Syk , Linfocitos T/metabolismo , Dominios Homologos src/fisiologíaRESUMEN
Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok-3 (downstream of kinase-3) as a key component of negative feedback regulation in Ca2+ signaling from the B-cell antigen receptor. Dok-3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok-3 inhibits antigen receptor-induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Bruton's tyrosine kinase. This leads to diminished activation of phospholipase C-gamma2 and reduced production of soluble inositol trisphosphate. Hence, the Dok-3/Grb2 module is a membrane-associated signaling organizer, which orchestrates the interaction efficiency of Ca2+-mobilizing enzymes.