Figure 2.
PKCɛ-dependent phosphorylation of vimentin and regulation of vimentin association in the vesicular compartment. (A) PKCɛ null (−/−) and PKCɛRE cells were treated with PKC inhibitor BIM-I where indicated and whole-cell lysates of equal protein loading were resolved with 2D gel electrophoresis and transferred onto nitrocellulose. Serine-phosphorylation was detected with blotting using an anti-phospho-serine antibody (left-hand panels) followed by stripping and reprobing with anti-vimentin to detect the localisation of the endogenous proteins (right-hand panels). In addition to vimentin, the phospho-serine antibody detects an unidentified protein spot migrating at slightly higher molecular weight than vimentin (marked ‘*'). The phospho-serine signal corresponding to vimentin is indicated with arrows. (B) Biotinylated vesicles from BIM-I treated PKCɛRE cells were resuspended in a small volume of buffer and incubated in cytosol derived from PKCɛ−/− cells in the presence of ATP and GTP, to support PKC catalytic activity, or buffer alone as indicated for 1 h at 37°, followed by refractionation on an equilibrium gradient. Biotinylated proteins from each fraction were collected on streptavidin-Dynabeads, the bound proteins were subjected to Western blot analysis following SDS–PAGE electrophoresis. (C) Isolated vesicles from BIM-I treated PKCɛRE cells were resuspended in a small volume of buffer and incubated for 1 h at 37°C in buffer in the presence of ATP and GTP. The reaction was diluted into two volumes of ice cold HEPES-buffer and sedimented at 100 000 g to recover the remaining membrane-bound proteins. Vimentin was immunoprecipitated from the soluble fraction (IP) and the remaining soluble proteins were concentrated with TCA precipitation. Proteins from all three fractions were assayed by Western blot analysis to detect PKCɛ and vimentin. (D) Isolated vesicles from BIM-I treated PKCɛRE cells were resuspended to a small volume of buffer and incubated for 1 h at 37°C in buffer in the presence of ATP and GTP and BIM I where indicated. The reaction was diluted into two volumes of ice cold HEPES-buffer and sedimented at 100 000 g to recover the remaining membrane bound proteins; the remaining soluble proteins were concentrated with TCA precipitation. Proteins from both fractions were assayed with Western blot analysis to detect vimentin phosphorylation at specific sites. Vimentin distribution was detected by stripping and reprobing after phospho-antibody detection. A representative blot with vimentin anti-phosphoserine 38 is shown in (D). (E) The histogram shows quantification of the distribution of vimentin phosphorylated at the sites indicated in the membrane bound and soluble fractions in a representative experiment. (F) A representative blot with vimentin anti-phosphoserine 6 is shown.