Protein phosphorylation involves a complex network of interacting proteins that allow signals at the plasma membrane to be translated and delivered to various sites within the cell. Protein kinases are a critical part of this network. This project focuses on cAMP-dependent protein kinase (PKA), one of the simplest and best understood protein kinases. In addition to understanding the structure of PKA, it is essential to know how it is regulated and localized. The discovery of A Kinase Anchoring Proteins (AKAP's) revealed a family of proteins that target PKA to various sites within the cell and has opened a new dimension in our thinking about PKA. This project focuses on two newly discovered families of AKAP's that bind both the RI and RII subunits of PKA. Dual specific (D)-AKAP targets PKA to either mitochondria or ER while D-AKAP2 has a putative RGS domain. Our goals are to study the anchoring and targeting motifs and domains of D- AKAP1 and 2 at both the molecular and cellular level. The specific motifs will be defined using deletion and site specific mutagenesis and then characterized using NMR with P. Jennings (Core) and/or X-ray crystallography with N. Xuong (Core). Fluorescence anisotropy will be used to characterize dynamics and conformational changes. Our initial goal is to define the anchoring motifs in D/AKAP1 and 2 and to characterize their interactions with the dimerization/docking domains at the N-terminus of RIalpha and RIIbeta. The D-AKAP's will be compared with the AKAP's described previously by J. Scott. Our second goal is to define the anchoring domains within the context of the full length D-AKAP and to characterize the consequences of anchoring on PKA function. Structural analysis of these larger proteins will require both NMR and crystallography. Our final goal is to define the physiological importance of targeting. In addition to using mutagenesis to localize and define these motifs; we shall develop a set of GFP labeled proteins that will allow us to visualize targeting of these proteins within living cells. Working with S. Adams (Core) we shall use genetically altered GFP's to design interacting pairs of proteins that can be followed with fluorescence resonance energy transfer. Physiological partners of the D- AKAP's will be identified by confocal microscopy as well as electron microscopy. Brown adipose tissue (BAT) where RIIbeta and D-AKAP1 are both expressed at high levels and co-localize to the outer mitochondrial membrane will be developed as a cellular model for D-AKAP1 function. With M. Ellisman (Core) the mitochondria will be mapped at high resolution. The Physiological consequences of disrupting anchoring using peptides will be characterized.
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