Identifying critical non-catalytic residues that modulate protein kinase A activity.
BACKGROUND
Distal interactions between discrete elements of an enzyme are critical for communication and ultimately for regulation. However, identifying the components of such interactions has remained elusive due to the delicate nature of these contacts. Protein kinases are a prime example of an enzyme with multiple regulatory sites that are spatially separate, yet communicate extensively for tight regulation of activity. Kinase misregulation has been directly linked to a variety of cancers, underscoring the necessity for understanding intramolecular kinase regulation.


RESULTS
A genetic screen was developed to identify suppressor mutations that restored catalytic activity in vivo from two kinase-dead Protein Kinase A mutants in S. cerevisiae. The residues defined by the suppressors provide new insights into kinase regulation. Many of the acquired mutations were distal to the nucleotide binding pocket, highlighting the relationship of spatially dispersed residues in regulation.


CONCLUSIONS
The suppressor residues provide new insights into kinase regulation, including allosteric effects on catalytic elements and altered protein-protein interactions. The suppressor mutations identified in this study also share overlap with mutations identified from an identical screen in the yeast PKA homolog Tpk2, demonstrating functional conservation for some residues. Some mutations were independently isolated several times at the same sites. These sites are in agreement with sites previously identified from multiple cancer data sets as areas where acquired somatic mutations led to cancer progression and drug resistance. This method provides a valuable tool for identifying residues involved in kinase activity and for studying kinase misregulation in disease states.
Kennedy
Eileen J
EJ
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.
Yang
Jie
J
Pillus
Lorraine
L
Taylor
Susan S
SS
Ghosh
Gourisankar
G
D050539:3-Isopropylmalate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013489:Suppression, Genetic
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D050539:3-Isopropylmalate Dehydrogenase; C524993:LEU2 protein, S cerevisiae; D011494:Protein Kinases; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C517758:Prkaca protein, mouse; C457179:TPK2 protein, S cerevisiae