M-PHASE-SPECIFIC PHOSPHORYLATION AND STRUCTURAL REARRANGEMENT OF THE CYTOPLASMIC CROSS-LINKING PROTEIN PLECTIN INVOLVE P34(CDC2) KINASE

Plectin, a widespread and abundant cytoskeletal cross-linking protein, serves as a target for protein kinases throughout the cell cycle, wit hout any significant variation in overall phosphorylation level. One o f the various phosphorylation sites of the molecule was found to be ph osphorylated preferentially during mitosis. By in vivo phosphorylation of ectopically expressed plectin domains in stably transfected Chines e hamster ovary cells, this site was mapped to the C-terminal repeat 6 domain of the polypeptide. The same site has been identified as an in vitro target for p34(cdc2) kinase. Mitosis-specific phosphorylation o f plectin was accompanied by a rearrangement of plectin structures, ch anging from a filamentous, largely vimentin-associated state in interp hase to a diffuse vimentin-independent distribution in mitosis as visu alized by immunofluorescence microscopy. Subcellular fractionation stu dies showed that in interphase cells up to 80% of cellular plectin was found associated with an insoluble cell fraction mostly consisting of intermediate filaments, while during mitosis the majority of plectin (> 75%) became soluble. Furthermore, phosphorylation of purified plect in by p34(cdc2) kinase decreased plectin's ability to interact with pr eassembled vimentin filaments in vitro. Together, our data suggest tha t a mitosis-specific phosphorylation involving p34(cdc2) kinase regula tes plectin's cross-linking activities and association with intermedia te filaments during the cell cycle.