Screening lytic phage display libraries for proteins which interact withcAMP-dependent protein kinase regulatory subunit type I-alpha

Cyclic AMP-dependent protein kinase (PKA) is an essential signaling element involved in the regulation and modulation of cardiac function. It is an integral part of several signaling pathways. Evidence is mounting showing the necessity for subcellular localization of PKA regulatory subunits (R) for linking effector to response. Co-localization of PKA with specific substrates streamlines signaling while maintaining agonist specificity. Discerning which PKA actions require type-specific localization and how localization is effected is important for a better understanding of the multiple roles PKA plays in the heart.; RII is localized through interaction of its dimerization domain with A kinase anchor proteins (AKAPs). While the localization of RIα is well established, the mode of RIα localization in the heart has not been elucidated. This study utilized a library screening technique called phage display in attempts to identify a RIα specific AKAP. Phage display is a powerful tool that allows the rapid screening of an entire cDNA library for protein-protein interacting pairs. Two lytic phage display systems were utilized in this study, Lambda D and T7 Select® (Novagen). A full length cDNA library was expressed in the Lambda D system. This library was screened against RIα and RIIα immobilized on CAMP affinity resin. No R subunit interacting proteins were identified in the screenings. A protein domain library was expressed in the T7 system and screened against RIα immobilized on cAMP affinity resin or Ni2+-NTA agarose. The T7 system was not able to identify a protein that interacts with RIα.