Detailed analysis of the phosphorylation of the human La (SS-B) autoantigen. (De)phosphorylation does not affect its subcellular distribution.
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.
Broekhuis
C H
CH
Department of Biochemistry, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands.
Neubauer
G
G
van der Heijden
A
A
Mann
M
M
Proud
C G
CG
van Venrooij
W J
WJ
Pruijn
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000235:genetics; D010449:Peptide Mapping; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis / Q000235:genetics; D000097585:SS-B Antigen
D001324:Autoantigens; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen