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  A 5′-fluorosulfonylbenzoyladenosine-based method to identify physiological substrates of a Drosophila p21-activated kinase

Menzel, N., Chari, A., Fischer, U., Linder, M., & Raabe, T. (2007). A 5′-fluorosulfonylbenzoyladenosine-based method to identify physiological substrates of a Drosophila p21-activated kinase. Analytical Biochemistry, 368(2), 178-184. doi:10.1016/j.ab.2007.05.022.

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Menzel, N., Author
Chari, A.1, Author           
Fischer, U., Author
Linder, M., Author
Raabe, T., Author
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1Research Group of Structural Biochemistry and Mechanisms, MPI for Biophysical Chemistry, Max Planck Society, ou_3265855              

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Free keywords: Identification of kinase phosphorylation substrates, Drosophila S2 cells, 5′FSBA inhibition of kinases, p21-activated kinase, Dynamitin
 Abstract: Nearly all processes in cells are regulated by the coordinated interplay between reversible protein phosphorylation and dephosphorylation. Therefore, it is a great challenge to identify all phosphorylation substrates of a single protein kinase to understand its integration into intracellular signaling networks. In this work, we developed an assay that holds promise as being useful for the identification of phosphorylation substrates of a given protein kinase of interest. The method relies on irreversible inhibition of endogenous kinase activities with the ATP analogue 5′-fluorosulfonylbenzoyladenosine (5′FSBA). 5′FSBA-treated cell extracts are then combined with a purified activated kinase to allow phosphorylation of putative substrate proteins, followed by a two-step purification protocol and identification by fingerprint mass spectrometry. Specifically, we applied this method to identify new phosphorylation substrates of the Drosophila p21-activated kinase (PAK) protein Mbt. Among candidate proteins identified by mass spectrometry, the dynactin complex subunit dynamitin was verified as a bona fide Mbt phosphorylation substrate and interaction partner, suggesting an involvement of this PAK protein in the regulation of dynactin-dependent cellular processes.

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Language(s): eng - English
 Dates: 2007-05-262007-09-15
 Publication Status: Published in print
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 Rev. Type: Peer
 Identifiers: DOI: 10.1016/j.ab.2007.05.022
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Title: Analytical Biochemistry
Source Genre: Journal
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Pages: - Volume / Issue: 368 (2) Sequence Number: - Start / End Page: 178 - 184 Identifier: -