Using FRET to visualize the anchoring of PKA in vivo

Peter Carmichael

Appointment Period: 1998-1999, Grant Year: [14]

My research involved the use of FRET to visualize the anchoring of PKA in vivo. Protein Kinase A (PKA) mediates a variety of hormonal and neurotransmitter responses within a cell. It consists of two catalytic subunits bound to two dimerized regulatory subunits. Upon stimulation with cAMP, the catalytic subunits are released from the holoenzyme, whereupon they can phosphorylate a variety of important serine/threonine substrates.

The localization of PKA to different subcellular compartments is important to its function. The Taylor lab has identified a new PKA anchoring protein known as Dual A Kinase Anchoring Protein (D-AKAP1) which is thought to localize the regulatory domain of PKAII to the mitochondria or endoplasmic reticulum (depending on the splice variant). My work involved a collaboration between Dr. Taylor and Dr. Roger Tsien, who has pioneered new techniques for visualizing protein binding within living cells. I am making fusions of the RII subunit of PKA with CFP (Cyan Fluorescent Protein) and D-AKAP1 with YFP (Yellow Fluorescent Protein). This allowed visualization of the localization of these proteins in single cells, using FRET to quantitate binding between them.

PUBLICATIONS (resulting from this training)

Sastri M, Barraclough DM, Carmichael PT, Taylor SS. (2005) A-kinase-interacting protein localizes protein kinase A in the nucleus. Proc Natl Acad Sci USA 102:349-54.