Abstract
We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. Here we report a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice displayed an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain.
Highlights
We have previously demonstrated that Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling
Microtubule regeneration assays revealed that Fes aggregates colocalized with ␥-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocked normal centrosome formation
Our findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain
Summary
Materials—Aprotinin, phenylmethylsulfonyl fluoride, phalloidinTRITC, anti-acetylated tubulin Ab, anti-␣-tubulin Ab, anti-␥-tubulin Ab, and anti-FLAG rabbit polyclonal Ab were purchased from Sigma. Anti-phosphotyrosine monoclonal Ab (4G10) and anti-Fes rabbit polyclonal Ab were from Upstate Biotechnology. Immunoprecipitates were boiled with SDS-PAGE sample buffer for 3 min, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore), followed by detection with the appropriate Ab as described previously [9]. Expression Constructs—Cloning of mouse Fes cDNA was performed as described previously [5]. Fes cDNA tagged with FLAG epitope at the N terminus were subcloned into pCMV5 expression vectors. Fes kinasenegative mutant was generated by missense mutation K562R as described previously [10]. Point mutation of Arg482 of Fes cDNA in SH2 domain to Lys (R482K) was performed by the site-directed mutagenesis kit from Strategene.
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