| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 89 === Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) is involved in p38 MAPK pathway, can phosphorylate vimentin. Previous studies showed that phosphorylation sites were on serine in the head of vimentin. In order to delineate the in vitro relationship of phosphorylation sites and assembly under MAPKAP kinase-2 treatment, serine substituted vimentin were constructed via site-directed mutagenesis of human vimentin cDNA and expressed in E. coli. We examined the assembly competence of recombinant vimentin and found that his-tagged vimentin would form aberrant filaments, whereas non-tagged vimentin could assembly into normal filaments. Using an expression system which produced the target protein without tag, we obtained assembly-competent wild-type and respective mutated vimentin. The effects of MAPKAP kinase-2 on the assembly competence were examined by centrifugation and negative staining electron microscopy. The experimental results showed that MAPKAP kinase-2-phosphorylated wild-type vimentin still retained the ability of forming filament, however, with wider diameter. Interestingly,mutation in Ser-55 did show normal assembly competence under MAPKAP kinase-2 treatment. It is possible that Ser-55 plays an important role in assembly of MAPKAP kinase-2- phosphorylated vimentin.
|
|---|
