RESUMO
Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.
Assuntos
Exodesoxirribonucleases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Quinase C/metabolismo , Interferência de RNA , RecQ Helicases/metabolismo , Receptores de Superfície Celular/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Dano ao DNA , Ativação Enzimática , Exodesoxirribonucleases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/genética , Humanos , Proteínas de Neoplasias/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteína Quinase C beta , Proteína Quinase C-delta/metabolismo , RecQ Helicases/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Helicase da Síndrome de Werner , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The ability of nonpathogenic Fusarium oxysporum, strain Fo47, to trigger plant defense reactions was investigated using Ri T-DNA-transformed pea roots. Cytological investigations of strain Fo47-inoculated roots showed that the fungus grew actively at the root surface and colonized a number of epidermal and cortical cells, inducing marked host cell metabolic changes. In roots inoculated with pathogenic F. oxysporum f. sp. pisi, the pathogen multiplied abundantly through much of the tissues, whereas in Fo47-inoculated roots, fungal growth was restricted to the epidermis and the outer cortex. Invading cells of strain Fo47 suffered from serious alterations, a phenomenon that was not observed in control roots in which F. oxysporum f. sp. pisi grew so actively that the vascular stele was invaded within a few days. Strain Fo47 establishment in the root tissues resulted in a massive elaboration of hemispherical wall appositions and in the deposition of an electron-opaque material frequently encircling pathogen hyphae and accumulating in the noninfected xylem vessels. This suggests that the host roots were signaled to defend themselves through the rapid stimulation of a general cascade of nonspecific defense responses. The specific relationship established between strain Fo47 and the root tissues is discussed in relation to other types of plant-fungus interactions, including pathogenic and symbiotic associations.
