RESUMEN
In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.
Asunto(s)
Apoptosis/fisiología , Autoantígenos/genética , Autoantígenos/inmunología , Autoantígenos/metabolismo , ARN/biosíntesis , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Anisomicina/farmacología , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Autoantígenos/efectos de los fármacos , Inhibidores de Caspasas , Caspasas/fisiología , Técnicas de Cultivo de Célula , Dactinomicina/farmacología , Dexametasona/farmacología , Inhibidores Enzimáticos/farmacología , Datos de Secuencia Molecular , Ácido Ocadaico/farmacología , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , Ribonucleoproteínas/efectos de los fármacos , Rayos Ultravioleta , Antígeno SS-BRESUMEN
The La (SS-B) autoantigen is an evolutionarily conserved phosphoprotein which plays an important role, most likely as an RNA chaperone, in various processes, such as the biosynthesis and maturation of RNA polymerase III transcripts in the cell nucleus and (internal) initiation of translation in the cytoplasm. In this study, the phosphorylation state of this protein from human HeLa and HEp-2 cells was characterized by high-resolution two-dimensional IEF/SDS-PAGE analysis, and phosphorylation sites were mapped by nanoelectrospray mass spectrometry. Furthermore, the effect of phosphorylation at the sites identified on the subcellular distribution of the protein was studied by site-directed mutagenesis. At least 14 isoelectric isoforms were discerned on 2-D gels with La protein from both types of cells. Metabolic labeling in combination with alkaline phosphatase treatment revealed that only a limited number of these isoforms could be attributed to phosphorylation. Four phosphorylation sites, Thr-302, Ser-325, Thr-362, and Ser-366, were mapped by mass spectrometric analysis of the isolated La protein from HeLa cells or the carboxy-terminal half of this protein. The analysis of mutants of La, in which the respective phosphorylated residues were replaced by either a neutral (alanine) or an acidic (aspartate) residue, by microinjection into Xenopus laevis oocytes on the one hand and transfection of HEp-2 cells on the other hand revealed that the subcellular distribution of this protein was not affected by these amino acid substitutions. These results strongly suggest that the signals that determine the subcellular distribution of this protein are not regulated by (de)phosphorylation of the target residues examined.