Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange

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題名:	Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange
作者:	Hsu, Y.-H.a, Johnson, D.A.b, Traugh, J.A.a
貢獻者:	Department of Chemistry, Tunghai University
日期:	2008
上傳時間:	2013-06-11T09:06:12Z (UTC)
摘要:	During apoptotic stress, protein kinase Pak2 is cleaved by caspase 3 to form a heterotetramer that is constitutively activated following autophosphorylation. The active protein kinase migrates slightly slower than the inactive holoenzyme when analyzed by gel filtration, suggesting an expanded conformation. Activation of Pak2 comprises a series of structural changes resulting from caspase cleavage, ATP binding, and autophosphorylation of Pak2. Changes at each step were individually analyzed by amide hydrogen/deuterium exchange coupled with mass spectrometry and compared with inactive Pak2. The autoinhibited form was shown to bind ATP in the active site, with minor changes in the glycine loop and the autoinhibitory domain (AID). Caspase cleavage produced significant changes in solvent accessibility in the AID and upper lobe of the catalytic domain. Cleavage of ATP-bound Pak2 relaxes the allosteric inhibition, as shown by increased solvent accessibility in the upper and lower lobes, including the G-helix, facilitating the autophosphorylation of two sites required for activation, Ser-141 in the regulatory domain and Thr-402 in the catalytic domain. Autophosphorylation increased the amide hydrogen/deuterium exchange solvent accessibility of the contact region between the AID and the G-helix, the E-F loop, and the N-terminus. Thus, activation of Pak2 via caspase cleavage is associated with structural relaxation of Pak2 that allows for complete autophosphorylation, resulting in a more comprehensive solvent-exposed and conformationally dynamic enzyme. ? 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
關聯:	Journal of Biological Chemistry Volume 283, Issue 52, 26 December 2008, Pages 36397-36405
顯示於類別:	[化學系所] 期刊論文

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