Detailed Analysis of the Phosphorylation of the Human La (SS-B) Autoantigen. (De)phosphorylation Does Not Affect Its Subcellular Distribution
详细信息 查看全文
- 作者：Christel H. D. Broekhuis ; Gitte Neubauer ; Annemarie van der Heijden ; Matthias Mann ; Christopher G. Proud ; Walther J. van Venrooij ; and Ger J. M. Pruijn
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：March 21, 2000
- 年：2000
- 卷：39
- 期：11
- 页码：3023 - 3033
- 全文大小：245K
- 年卷期：v.39,no.11(March 21, 2000)
- ISSN：1520-4995
文摘
The La (SS-B) autoantigen is an evolutionarily conserved phosphoprotein which plays animportant role, most likely as an RNA chaperone, in various processes, such as the biosynthesis andmaturation of RNA polymerase III transcripts in the cell nucleus and (internal) initiation of translation inthe cytoplasm. In this study, the phosphorylation state of this protein from human HeLa and HEp-2 cellswas characterized by high-resolution two-dimensional IEF/SDS-PAGE analysis, and phosphorylationsites were mapped by nanoelectrospray mass spectrometry. Furthermore, the effect of phosphorylation atthe 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 numberof 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 HeLacells or the carboxy-terminal half of this protein. The analysis of mutants of La, in which the respectivephosphorylated residues were replaced by either a neutral (alanine) or an acidic (aspartate) residue, bymicroinjection into Xenopus laevis oocytes on the one hand and transfection of HEp-2 cells on the otherhand revealed that the subcellular distribution of this protein was not affected by these amino acidsubstitutions. These results strongly suggest that the signals that determine the subcellular distribution ofthis protein are not regulated by (de)phosphorylation of the target residues examined.