Detailed Analysis of the Phosphorylation of the Human La (SS-B) Autoantigen. (De)phosphorylation Does Not Affect Its Subcellular Distribution

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.

Cleavage of La protein by granzyme H induces cytoplasmic translocation and interferes with La-mediated HCV-IRES translational activity

“…In addition, it contains a multitude of closely associated and sometimes overlapping functional and posttranslational regulatory regions (Figure 2b), including nuclear and nucleolar localization signals and multiple phosphorylation sites (i.e., Thr-302, Ser-325 Thr-362, and Ser-366). 22,25,37 Interestingly, gzmH cleaves La at the C-terminal region, and its cleavage efficiency depends on unique features found in this domain. Thus, the presence of a track of negatively charged residues C-terminal to the scissile bond (from P 3 0 to P 5 0 ) ensures efficient cleavage, likely by promoting efficient interaction of gzmH with La through the formation of a salt bridge with Lys-27 in gzmH.…”

Sumoylation in axons triggers retrograde transport of the RNA-binding protein La

“…La can be phosphorylated (16), but the number of phosphates needed for a 20-kDa shift would substantially alter La's pI, and such was not seen (SI Fig. 8).…”

Akt phosphorylation of La regulates specific mRNA translation in glial progenitors

“…Akt phosphorylates La protein in vitro La can be phosphorylated on its C-terminus in vitro by several kinases such as casein kinase II, protein kinase A or protein kinase C (Broekhuis et al, 2000). As La is known to be a phosphoprotein, we determined whether the phosphorylation state of La was regulated by the PI3K-Akt pathway.…”