RESUMEN
alpha-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to alpha-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking alpha-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by alpha-thrombin. Further, although several Galpha subunits associate with PAR1 and are released following stimulation, only Galpha(i2) couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gbeta and Ggamma subunits, only a subset associates with Galpha(i2), and of those, only a single Gbetagamma dimer, Gbeta(1)gamma(5), participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by alpha-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking alpha-thrombin stimulation to the activation of Ras.
Asunto(s)
Fibroblastos/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Microdominios de Membrana/metabolismo , Trombina/farmacología , Proteínas ras/metabolismo , Animales , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Fibroblastos/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Humanos , Microdominios de Membrana/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation.