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KMID : 0390320110210010239
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Chungbuk Medical Journal
2011 Volume.21 No. 1 p.239 ~ p.244
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Regulation of PAK2 Phosphorylation by ERK Signal Transduction Pathway
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Kim Eung-Gook
Shin Eun-Young
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Abstract
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Purpose: P21-activated kinase (PAK2) was phosphorylated by activated extracellualr signalregulated kinase (ERK) in vitro. This study analyzed the four phosphorylation sites for PAK2 found in vitro kinase assay that were phosphoylated by ERK1/2 using unphophorylable mutant PAK2 constructs in cells.
Materials and Methods : To obtain unphosphorylable mutants for S32, T154, S197 and T510 sites of PAK2, myc-PAK2 (WT) or PAK2-K278R was mutated into alanine by Quickchange site directed mutagenesis using each specific mutant primer set. These PAK2 mutants were named with myc-PAK2-W-4M and myc-PAK2-K278R-4M, respectively. Plasmids for PAK2 mutants with active MEK were transfected on PC12 cells using LF2000 for 24 hr. Phosphorylation of PAK2 was confirmed by mobility shift on SDS-PAGE and Western blotting with anti-PAK2 antibody.
Results: Unphosphorylable mutant PAK2s were still phosphorylated by active MEK in cells in cells.
Conclusion: These results suggest that ERK1/2 phosphorylated the another sites of PAK2 except S32, T154, S197 and T510 sites, or PAK2 phosphorylation by ERK1/2 can be detected only in vitro.
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KEYWORD
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PAK2, ERK1/2, Mutagenesis, Phosphorylation
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