title: Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments.
abstract: Intermediate filaments (IFs) continuously exchange between a small, depolymerized fraction of IF protein and fully polymerized IFs. To elucidate the possible role of phosphorylation in regulating this equilibrium, we disrupted the exchange of phosphate groups by specific inhibition of dephosphorylation and by specific phosphorylation and site-directed mutagenesis of two of the major in vivo phosphorylation sites determined in this study. Inhibition of type-1 (PP1) and type-2A (PP2A) protein phosphatases in BHK-21 fibroblasts with calyculin-A, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This oligomeric composition corresponded to the oligopeptides released by cAMP-dependent kinase (PKA) following in vitro phosphorylation. Characterization of the (32)P-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser-7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser-429, Thr-456, and Ser-457 as significant in vivo phosphorylation sites. A number of the interphase-specific high turnover sites were shown to be in vitro phosphorylation sites for PKA and protein kinase C (PKC). The effect of presence or absence of phosphate groups on individual subunits was followed in vivo by microinjecting PKA-phosphorylated (primarily S38 and S72) and mutant vimentin (S38:A, S72:A), respectively. The PKA-phosphorylated vimentin showed a clearly decelerated filament formation in vivo, whereas obstruction of phosphorylation at these sites by site-directed mutagenesis had no significant effect on the incorporation rates of subunits into assembled polymers. Taken together, our results suggest that elevated phosphorylation regulates IF assembly in vivo by changing the equilibrium constant of subunit exchange towards a higher off-rate.
authors.affiliation: Department of Biology, Laboratory of Animal Physiology, University of Turku, Science Building 1, FIN-20014 Turku, Finland. john.eriksson@utu.fi
authors.forename: John E
authors.initials: JE
authors.lastname: Eriksson
authors.forename: Tao
authors.initials: T
authors.lastname: He
authors.forename: Amy V
authors.initials: AV
authors.lastname: Trejo-Skalli
authors.forename: Ann-Sofi
authors.initials: AS
authors.lastname: Härmälä-Braskén
authors.forename: Jukka
authors.initials: J
authors.lastname: Hellman
authors.forename: Ying-Hao
authors.initials: YH
authors.lastname: Chou
authors.forename: Robert D
authors.initials: RD
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D008387:Marine Toxins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010080:Oxazoles / Q000494:pharmacology*; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D012694:Serine / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D014746:Vimentin; D012694:Serine; C059041:calyculin A; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C