Abstract
Kinases function as regulators of many cellular processes such as cell migration. These enzymes typically phosphorylate target motif sequences. Mass spec or phospho-specific antibody detection can be used to determine whether a kinase can phosphorylate proteins of interest, however, mass spec can be expensive and phospho-antibodies for the protein of interest may not exist. In this protocol, we will describe an in vitro kinase assay to provide a preliminary readout on whether a protein of interest may be phosphorylated by PKA. Our protein of interest is purified after expression in bacteria and treated with recombinant PKA from bovine heart. Protein is then extracted and a western blot is performed using a phospho-specific antibody for PKA’s target motif. This will allow us to quickly determine if it is possible for PKA to phosphorylate our protein of interest.
Keywords: Protein Kinase A (PKA), Kinase activity, In vitro, Western blot, Protein phosphorylation
Background
Hormones and other factors that cause activation of adenylate cyclase through G-linked G-protein coupled receptors (GPCR) and subsequently promote generation of second messenger cAMP can affect cellular processes like cell migration. Elevation of cAMP level leads to activation of PKA, a serine-threonine kinase that plays an important role in the regulation of actin cytoskeletal dynamics in migrating cells. PKA influences different facets of actin cytoskeleton-regulatory processes including modulation activities of a) Rho-family GTPases (Rho, Rac and Cdc42), b) actin-binding proteins (e.g., VASP [vasodilator stimulated phosphoprotein]), c) kinases which indirectly control the function of actin-binding proteins (e.g., p21-activated kinase) and d) myosin (Howe, 2004). PKA and other kinases, however, can also modulate other proteins by phosphorylation that are currently unknown. There are multiple ways to determine kinase activity on a protein of interest such as mass spec for phosphopeptides, however, such assays can be costly. In this protocol, we will describe the use of an in vitro kinase assay to examine PKA kinase activity on a GST-tagged protein of interest using a phospho-specific antibody for PKA’s target motif. Using a PKA motif antibody is advantageous if known phosphorylation sites are unknown on a protein or if no phosphospecific protein antibody exists. While this assay is quick and simple to perform, it depends on the kinase targeting a motif pre-determined by available antibodies and thus limited in potential. A different kinase can be easily tested if an antibody to detect that kinase’s phospho motif exists. Despite this, successful readout from this assay can provide motivation to pursue more comprehensive exploration into kinase phosphorylation of a protein of interest. Although this protocol describes usage of recombinant protein purified from bacteria, protein can be obtained from any source (i.e., insect or human cells) but total yield of protein will vary dependent on the source and may need to be optimized. Figure 1 below illustrate the general workflow for this protocol.
Figure 1. General protocol workflow for kinase analysis
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This work was supported by a grant from the National Institute of Health (2R01CA108607) to PR. David Gau was supported by a National Science Foundation pre-doctoral fellowship (2012139050) and an NIH Cardiovascular Bioengineering pre-doctoral training grant (2T32HL076124 to DG).
Competing interests
The authors have no competing interests to report.
References
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