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  Phosphorylation and flexibility of cyclic-AMP-dependent protein kinase (PKA) using P-31 NMR Spectroscopy

Seifert, M. H. J., Breitenlechner, C. B., Bossemeyer, D., Huber, R., Holak, T. A., & Engh, R. A. (2002). Phosphorylation and flexibility of cyclic-AMP-dependent protein kinase (PKA) using P-31 NMR Spectroscopy. Biochemistry, 41(19), 5968-5977.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-6F32-5 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-6F33-3
Genre: Journal Article
Alternative Title : Biochemistry

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 Creators:
Seifert, M. H. J.1, Author              
Breitenlechner, C. B.1, Author              
Bossemeyer, D., Author
Huber, R.2, Author              
Holak, T. A.3, Author              
Engh, R. A.2, Author              
Affiliations:
1External Organizations, ou_persistent22              
2Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565155              
3Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565154              

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 Abstract: Cell signaling pathways rely on phosphotransfer reactions that are catalyzed by protein kinases. The protein kinases themselves are typically regulated by phosphorylation and concurrent structural rearrangements, both near the catalytic site and elsewhere. Thus, physiological function requires posttranslational modification and deformable strictures. A prototypical example is provided by cyclic AMP-dependent protein kinase (PKA). It is activated by phosphorylation, is inhomogeneously phosphorylated when expressed in bacteria, and exhibits a wide range of dynamic properties. Here we use P-31 nuclear magnetic resonance (NMR) spectroscopy to characterize the phosphorylation states and to estimate the flexibility of the phosphorylation sites of 2-, 3-, and 4-fold phosphorylated PKA. The phosphorylation sites Ser10, Ser139, Thr197, and Ser338 are assigned to individual NMR resonances, assisted by complexation with AMP-PNP and dephosphorylation with alkaline phosphatase. Rotational diffusion correlation times estimated from resonance line widths show progressively increasing flexibilities for phosphothreonine 197, phosphoserines 139 and 338, and disorder at phosphoserine 10, consistent with crystal structures of PKA. However, because the apparent rotational diffusion correlation time fitted for phosphothreonine 197 of the activation loop is longer than the overall PKA rotational diffusion time, microsecond to millisecond time scale conformational exchange effects involving motions of phosphothreonine 197 are probable. These may represent "open"- "closed" transitions of the uncomplexed protein in solution. These data represent direct measurements of flexibilities also associated with functional properties, such as ATP binding and membrane association, and illustrate the applicability of P-31 NMR for functional and dynamic characterization of protein kinase phosphorylation sites.

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Language(s): eng - English
 Dates: 2002-05-14
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 39339
ISI: 000175547000005
 Degree: -

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Title: Biochemistry
  Alternative Title : Biochemistry
Source Genre: Journal
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Pages: - Volume / Issue: 41 (19) Sequence Number: - Start / End Page: 5968 - 5977 Identifier: ISSN: 0006-2960