Fig. 1. Inhibition of vimentin dephosphorylation in vivo induces hyperphosphorylation and disassembly of vimentin polymers. (A) ³²P in vivo labeled BHK-21 cells were incubated without or with 20 nM of the protein phosphatase inhibitor cl-A for 10 and 20 minutes and with 50 nM cl-A for 30 minutes. The inhibition of constitutive vimentin phosphatase activities causes a dose and time-dependent elevation of vimentin phosphorylation, as shown in the autoradiography of the ³²P in vivo labeled proteins separated by 10% SDS-PAGE. (B) The phosphorylation causes a disassembly of the vimentin IF filaments, which is reflected by their increase in both the low and high speed centrifugation supernatants, as measured by western blotting of the respective supernatant fractions. While the IF pool in the low speed supernatant is primarily composed of both large filament fragments and soluble subunits, the high speed supernatant contains only truly soluble subunits. The vimentin levels are elevated both in the low and high speed supernatants but desmin concentrations are only elevated in the low speed supernatants. This implies that desmin is not disassembled under these conditions into soluble subunits but merely fragmented. This is consistent with the phosphorylation of desmin, which is not elevated by inhibition of dephosphorylation. (C) The specific phosphorylation (³²P labeling/protein units) of polymer-associated and depolymerized vimentin subunits was analyzed by double in vivo labeling with [³²P]orthophosphate and [³⁵S]methionine followed by phosphorimager analysis (³²P + ³⁵S is the signal collected from both isotopes on the gel; the ³²P signal was obtained by exposure through four layers of aluminum foil). The results indicate that phosphate incorporation takes place both on polymer-associated vimentin and on the dissociated subunits, as shown by (D) quantification of the same specific levels of ³²P per protein unit (³²P/³⁵S), with a certain preference for phosphate incorporation into the soluble subunits, as indicated by the ratios between the different protein pools, as compared to the filamentous pool (=1), shown above the bars.