Objective: To monitor the phosphorylation modifications and cellular localization of poly(rC)-binding protein-1 (PCBP1) during the cell cycle progression of Hela cells.
Result: Hela cells highly synchronized at five different phases from interphase to mitosis were obtained. Using mitotic phosphoprotein-specific monoclonal antibody MPM-2, the exclusive occurrences of multiphosphorylation statuses of PCBP1 in mitosis were confirmed by a series of spots with increasing acidic pI (isoelectric point) in two rounds of 2D western blotting on the same membrane, and a visible molecular mass shift that can be eliminated by the treatment with λ phosphatase in 1D western blotting. Immnuofluorescence revealed the localization shift of PCBP1 during cell cycle, with accumulations in nucleus as a patch pattern in interphase, and a dispersive distribution without the area of the condensed chromosomes during mitosis.
Conclusions: These observations of mitosis-specific multiphosphorylations and localization shifts of PCBP1 suggest that the versatile PCBP1 was regulatable in a phosphorylation modification- and temporospatial-dependent manner in mitotic regulatory networks.
Keywords: Cell cycle; Localization; MPM-2; PCBP1; Phosphorylation; Poly(rC)-binding protein.