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First published online 3 February 2004
doi: 10.1242/jcs.00906
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Research Article |
1 Department of Biology, Laboratory of Animal Physiology, University of Turku, Science Building 1, FIN-20014 Turku, Finland
2 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, POB 123, FIN-20521 Turku, Finland
3 Department of Biochemistry, Åbo Akademi University, FIN-20521 Turku, Finland
4 Turku Graduate School of Biomedical Sciences, Kiinanmyllynkatu 13, FIN-20520, Turku, Finland
5 Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, 303 E. Chicago Avenue, Chicago, IL-60611-3008, USA
* Author for correspondence (e-mail: john.eriksson{at}utu.fi)
Accepted 29 September 2003
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 32P-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.
Key words: Phosphorylation, Signaling, Intermediate filaments, Cytoskeleton, Dynamics
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