Cite as: Cold Spring Harb. Protoc.; 2007; doi:10.1101/pdb.prot4638

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Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Kinase-Associated Substrates

Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber

This protocol was adapted from "Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates," Chapter 24, in Protein-Protein Interactions (ed. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.


INTRODUCTION

This protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [{gamma}-32P]ATP analog. This technique has been used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates; however, the methodology can be applied to other protein kinases as well.


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Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [{gamma}-32P]ATP Analog from ADP Analog
Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber
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Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Substrates in Cell Lysates with Exogenous Kinase
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CSH Protocols 2007: 4639. [Abstract] [Full Text]



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