| Autor: |
Eriksson, John E., He, Tao, Trejo-Skalli, Amy V., Härmälä-Braskén, Ann-Sofi, Hellman, Jukka, Chou, Ying-Hao, Goldman, Robert D. |
| Předmět: |
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| Zdroj: |
Journal of Cell Science; 2/29/2004, Vol. 117 Issue 6, p919-932, 14p, 3 Color Photographs, 12 Black and White Photographs, 2 Charts, 9 Graphs |
| Abstrakt: |
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 calyculinA, induced rapid vimentin phosphorylation in concert with disassembly of the IF polymers into soluble tetrameric vimentin oligomers. This ofigomeric composition corresponded to the oligopeptides released by cAMPdependent kinase (PKA) following in vitro phosphorylation. Characterization of the 32p-labeled vimentin phosphopeptides, demonstrated Ser-4, Ser-6, Ser7, Ser-8, Ser-9, Ser-38, Ser-41, Ser-71, Ser-72, Ser-418, Ser429, 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 $38 and $72) and mutant vimentin ($38:A, $72:A), respectively. The PKAphosphorylated 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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