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KMID : 0390320080180010322
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Chungbuk Medical Journal
2008 Volume.18 No. 1 p.322 ~ p.329
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Actin cytoskeletal rearrangement by PAK-mediated RhoGDI phosphorylation
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Kim Eung-Gook
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Abstract
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Purpose: P21-activated kinase (PAK) regulates actin dynamics via multiple downstream effectors. PAK forms a complex with PAK interacting exchange factor (PIX), Rac and RhoGDI. RhoGDI and PIX have been identified as downstream effectos. We therefore explored the effect of PAK-mediated RhoGDI phosphorylation on actin dynamics.
Materials & Method: RhoGDI was cloned into pGEX vector and expressed as GST-RhoGDI fusion proteins in E. coli. GST-RhoGDI was purified by glutathione affinity chromatography. Mutagenesis on RhoGDI phosphorylation sites was conducted using a mutagenesis kit and phosphorylation of Rho-GDI by active PAK was carried out by incubation with kinase reaction buffer containing 500 uM ATP at 30℃ for 30 min. Actin cytoskeletal rearrangement by wild-type or mutant RhoGDIs was determined by immunocytochemistry.
Results: We found that the S101 of RhoGDI is a major phosphorylation site. Its phosphomimetic mutant form, S101E/S174E (EE mutant), induced membrane ruffles. In contrast, wild-type and nonphosphorylatable mutant (S101A/S174A, AA mutant) did not induce any significant changes on cytoskeletal rearrangement compared to control GEF-transfected cells.
Conclusions: PAK may regulate actin dynamics through phosphorylation of RhoGDI.
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KEYWORD
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RhoGDI, PAK, Phosphorylation, Actin dynamics
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