RESUMEN
Activation of mitogen-activated protein kinases (MAPK) is a critical signal transduction event for CTL activation, but the signaling mechanisms responsible are not fully characterized. Protein kinase C (PKC) is thought to contribute to MAPK activation following TCR stimulation. We have found that dependence on PKC varies with the method used to stimulate the T cells. Extracellular signal-regulated kinase (ERK) activation in CTL stimulated with soluble cross-linked anti-CD3 is completely inhibited by the PKC inhibitor bisindolylmaleimide (BIM). In contrast, only the later time points in the course of ERK activation are sensitive to BIM when CTL are stimulated with immobilized anti-CD3, a condition that stimulates CTL degranulation. Surprisingly, MAPK activation in response to immobilized anti-CD3 is strongly inhibited at all time points by the diacylglycerol (DAG)-binding domain inhibitor calphostin C implicating the contribution of a DAG-dependent but PKC-independent pathway in the activation of ERK in CTL clones. Chronic exposure to phorbol ester down-regulates the expression of DAG-responsive PKC isoforms; however, this treatment of CTL clones does not inhibit anti-CD3-induced activation of MAPK. Phorbol ester-treated cells have reduced expression of several isoforms of PKC but still express the recently described DAG-binding Ras guanylnucleotide-releasing protein. These results indicate that the late phase of MAPK activation in CTL clones in response to immobilized anti-CD3 stimulation requires PKC while the early phase requires a DAG-dependent, BIM-resistant component.
Asunto(s)
Diglicéridos/metabolismo , Activación de Linfocitos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/fisiología , Linfocitos T Citotóxicos/enzimología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos/fisiología , Degranulación de la Célula/efectos de los fármacos , Células Clonales , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Interleucina-2/fisiología , Maleimidas/farmacología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 3 Activada por Mitógenos , Muromonab-CD3/metabolismo , Muromonab-CD3/farmacología , Naftalenos/farmacología , Fosforilación , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Proteína Quinasa C/antagonistas & inhibidores , Estructura Terciaria de Proteína/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Requirements for T cell activation are not fully established. One model is that receptor occupancy and down-regulation are essential for activation, and another, not necessarily mutually exclusive, model is that sustained signals are important. Here we examine the importance of signal duration in T cell activation. First, we demonstrate that immobilized, but not soluble cross-linked, Abs to CD3 stimulate degranulation by CTL. The cross-linked Abs are not deficient in their ability to signal since they stimulate the same tyrosine phosphorylation pattern as immobilized Ab, but it is very transient relative to that stimulated by immobilized Ab. Furthermore, novel decreased migratory forms of Lck occur to a significant extent only after stimulation with immobilized Abs. A dramatic difference in the duration of signals is very evident when mitogen-activated protein kinase (MAPK) activity is examined. Immobilized anti-CD3 stimulates very high levels of MAPK activation that is still detectable 1 h after stimulation. In contrast, cross-linked Ab stimulates only transient and incomplete activation of MAPK. Taken together, these results suggest that TCR engagement and induction of tyrosine phosphorylation alone are not sufficient for T cell activation and that the duration of TCR-stimulated signals is critical to attain a functional response.