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. 2013 Sep 15;24(18):2990–2999. doi: 10.1091/mbc.E13-04-0204

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© 2013 Shin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — CK2 phosphorylates PAK1 on S223. (A) Mass spectrometry identification of CK2-catalyzed PAK1 phosphorylation at S223. The 293T cells treated with a nontargeting shRNA (NT, top) or a CK2α-specific shRNA (shRNA, bottom) were infected with lentivirus expressing GFP-PAK1. GFP-PAK1 was immunoprecipitated with anti-GFP antibody, resolved by SDS–PAGE, and digested with trypsin or chymotrypsin/elastase. The resulting eluted peptides were analyzed directly by LC-MS/MS. The m/z difference between y-13s (arrows) of the peptides derived from 293T cells with (bottom) or without (top) CK2α knockdown indicates a phosphoserine at position 223. (B) Effect of CK2α knockdown on PAK1 phosphorylation in vivo. Western blot analysis of expression of GFP-PAK1-KD, CK2α, and actin in 293T cells with (sh1 and sh2) or without (NT) CK2α knockdown (WB). The 293T cells expressing GFP-PAK1-KD were metabolically labeled with [³²P]orthophosphate. GFP-PAK1-KD was immunoprecipitated with anti-GFP antibody and analyzed by autoradiography. (C) Effect of the CK2 inhibitor TBB on PAK1 phosphorylation in vivo. The 293T cells expressing GFP-PAK1-KD were treated with different concentrations of TBB, as indicated above each lane, and metabolically labeled with [³²P]orthophosphate. (D) In vitro phosphorylation of PAK1 by CK2. Bacterially expressed GST-PAK1-KD or GST-PAK1-KD-S223A was incubated with purified CK2 holoenzyme (20 ng) and [³²P]γ-ATP and analyzed by autoradiography. (E) Effect of the CK2 inhibitor heparin on PAK1 phosphorylation in vitro. Bacterially expressed GST-PAK1-KD was incubated with purified CK2 holoenzyme (20 ng) and [³²P]γ-ATP in the absence and presence of different amounts of heparin (lane 3: 25 μg; lane 4: 5 μg; lane 5: 1 μg) for 30 min and analyzed by autoradiography.