RESUMEN
A cDNA encoding a novel, human, dual-specific protein phosphatase was identified in the Incyte data base. The open reading frame predicted a protein of 184 amino acids related to the Vaccinia virus VH1 and human VH1-related (VHR) phosphatases. Expression VHR-related MKPX (VHX) was highest in thymus, but also detectable in monocytes and lymphocytes. A VHX-specific antiserum detected a protein with an apparent molecular mass of 19 kDa in many cells, including T lymphocytes and monocytes. VHX expression was not induced by T cell activation, but decreased somewhat at later time points. In vitro, VHX dephosphorylated the Erk2 mitogen-activated protein kinase with faster kinetics than did VHR, which is thought to be specific for Erk1 and 2. When expressed in Jurkat T cells, VHX had the capacity to suppress T cell antigen receptor-induced activation of Erk2 and of an NFAT/AP-1 luciferase reporter, but not an NF-kappaB reporter. Thus, VHX is a new member of the VH1/VHR group of small dual-specific phosphatases that act in mitogen-activated protein kinase signaling pathways.
Asunto(s)
Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Represoras , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Complementario/metabolismo , Fosfatasa 3 de Especificidad Dual , Fosfatasas de Especificidad Dual , Glutatión Transferasa/metabolismo , Humanos , Immunoblotting , Células Jurkat , Ligandos , Luciferasas/metabolismo , Linfocitos/enzimología , Sistema de Señalización de MAP Quinasas , Ratones , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Linfocitos T/metabolismo , Timo/enzimología , Factores de Tiempo , Distribución Tisular , Transfección , Vaccinia/enzimologíaRESUMEN
In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser(364) by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.