This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein Kinase A (PKA) is a central intracellular kinase that regulates the activity of many proteins involved in cellular metabolism. PKA activity is controlled via interactions with A Kinase Anchoring Proteins (AKAPs). AKAPs function by binding to the PKA regulatory subunit, localizing PKA within the cell. AKAPs can interact with either the αor the βisoform of the regulatory subunit of PKA, or they can interact with both. The αand βisoforms are highly conserved, making it difficult to study the molecular determinants of selectivity between isoforms. We are using phage display in combination with high-throughput sequencing to identify the sequence determinants of AKAP selectivity. We displayed a library of millions of mutagenized AKAP proteins on the surface of T7 phage and then subjected this library to selection against either the αor βisoform of the regulatory subunit of PKA. By comparing the abundance of each variant before and after selection, we derived enrichment ratios for several hundred thousand variants. Most variants performed similarly in selections against both the αand βisoforms. However, some variants displayed strong selectivity for either the αor βisoform. We are using the results of this assay in an effort to develop highly α- and β-specific AKAPs, which should bind only to PKAs with the cognate regulatory isoform. If introduced into cells at high concentrations, they should disrupt the normal regulatory interaction for their cognate isoform, enabling us to study the biological significance of the isoforms

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
2P41RR011823-16
Application #
8365785
Study Section
Special Emphasis Panel (ZRG1-CB-L (40))
Project Start
2011-09-01
Project End
2012-06-30
Budget Start
2011-09-01
Budget End
2012-06-30
Support Year
16
Fiscal Year
2011
Total Cost
$21,815
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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