The reverse in-gel kinase assay to profile physiological kinase substrates.
Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the alpha catalytic subunit of protein kinase CK2 (CK2alpha) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2alpha and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.
Li
Xiang
X
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
Guan
Bin
B
Srivastava
Minu K
MK
Padmanabhan
Achuth
A
Hampton
Brian S
BS
Bieberich
Charles J
CJ
D000180:Acrylic Resins / Q000737:chemistry*; D000818:Animals; D047390:Casein Kinase II / Q000737:chemistry / Q000378:metabolism; D002364:Caseins / Q000737:chemistry; D002384:Catalysis; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D008297:Male; D051379:Mice; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D010766:Phosphorylation; D000071876:Prostaglandin-E Synthases; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D012669:Seminal Vesicles / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014230:Triazoles / Q000737:chemistry
C405354:4,5,6,7-tetrabromobenzotriazole; D000180:Acrylic Resins; D002364:Caseins; D002457:Cell Extracts; D014230:Triazoles; C016680:polyacrylamide gels; D011494:Protein Kinases; D047390:Casein Kinase II; D017868:Cyclic AMP-Dependent Protein Kinases; D010744:Phosphoric Monoester Hydrolases; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases