Protein arginylation is a posttranslational modification mediated by arginyl-tRNA protein transferase (Ate 1) that transfers arginine from tRNA onto proteins that expose Glu, Asp or Cys at the N-terminal position. Whilst arginylation was discovered more than 40 years ago, the identity of proteins arginylated in vivo and its biological functions are largely unknown. We demonstrated the posttranslational incorporation of arginine into calreticulin (CRT) in vitro and in living cells. The arginylated form of CRT (R-CRT) was observed in the citosol, in contrast to the non arginylated CRT which is in the endoplasmatic reticulum.
It has been previously demonstrated that CRT affects cell adhesion, and plays a role in the control of cell adhesiveness via regulation of vinculin expression. This may be mediated by direct interaction between CRT and the KxGFFKR of a integrins. Therefore, to functionally affect integrin cluster in focal contacts, CRT should be in the cytoplasm. The first direct evidence of cytosolic CRT is the arginylated form R-CRT.
By immunocytochemistry, using an antibody against R-CRT we observed an increased expression of both R-CRT and vinculin in cells subjected to focal adhesion disassembly and reassembly. In addition, by confocal microscopy we observed the colocalization between integrins and R-CRT in hippocampal neurons and fibroblasts. Further studies performed in cells lacking of arginyl-tRNA protein transferase (ate1 -/-) show a differential attachment to laminin and polylisine when compared to cells ate1 +/+ (provided by Dr. Kashina). When both cell types were transfected with CRT the ate1 -/- cells did not improve their adhesiveness to substrata as ate1 +/+ cells.
This evidence supports the idea that R-CRT is implicated in cell adhesion and spreading; thus posttranslational arginylation of CRT seems to regulate its intracellular localization and cytosolic function.
Supported by SECyT-UNC, CONICET, ANPCyT-PICT.
