Format

Send to

Choose Destination
Biochemistry. 2000 Mar 21;39(11):3023-33.

Detailed analysis of the phosphorylation of the human La (SS-B) autoantigen. (De)phosphorylation does not affect its subcellular distribution.

Author information

1
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands.

Abstract

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.

PMID:
10715123
DOI:
10.1021/bi992308c
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for American Chemical Society
Loading ...
Support Center