PERIPHERIN IS TYROSINE-PHOSPHORYLATED AT ITS CARBOXYL-TERMINAL TYROSINE

Peripherin is a type III intermediate filament present in peripheral a nd certain CNS neurons, We report here that peripherin contains a phos photyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specif ic for phosphotyrosine recognizes peripherin present in PC12 cells (wi th or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim(-) cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC1 2 cells was confirmed by immunoprecipitation, two-dimensional isoelect ric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phospho rylation, tyrosine phosphorylation of peripherin is not regulated by d epolarization or nerve growth factor treatment, To identify the site o f tyrosine phosphorylation, rat peripherin was mutated at several tyro sine residues and expressed in SW-13 ct. 2 vim(-) cells. Tyrosine phos phorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluor escence staining indicated that both wild-type peripherin and peripher in Y474F form a filamentous network in SW-13 cl. 2 vim(-) cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal r esidue is not required for assembly and leaves open the possibility th at this modification serves other functions.