A 5'-fluorosulfonylbenzoyladenosine-based method to identify physiological substrates of a Drosophila p21-activated kinase.
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.
Menzel
Nicolas
N
Institute of Medical Radiation and Cell Research, University of Würzburg, D-97078 Würzburg, Germany.
Chari
Ashwin
A
Fischer
Utz
U
Linder
Monica
M
Raabe
Thomas
T
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D000072159:Dynactin Complex; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D054462:p21-Activated Kinases / Q000737:chemistry / Q000378:metabolism*
D029721:Drosophila Proteins; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D011494:Protein Kinases; C099277:Pak protein, Drosophila; C116684:mbt protein, Drosophila; D054462:p21-Activated Kinases; D000241:Adenosine