Signaling crosstalk between protein kinase A and GTPases

The specificity of receptor initiated signaling responses is encoded by spatial and temporal dynamics of downstream signaling networks. These networks, initiating from e.g. the G protein coupled receptor (GPCR) superfamily and receptor tyrosine kinases, tightly regulate signaling pathways at several critical points via feedback loops and crosstalk among other pathways. Signaling networks are complex and the apparent crosstalk between different pathways is rationalized based on complicated models. However, crosstalk can often be explained by simple and occasionally surprising molecular interactions. Elucidation of the physical connection between these pathways is also a key to understand aberrant cell signaling. During my postdoctoral studies we have recognized that the protein-fragment complementation assay 1,25 based on the yellow fluorescent protein provides the opportunity to trap transient protein:protein interactions (PPI) in vivo. Therefore we set out to identify novel binary interactions which represent crosstalk and feedback loops between and within pathways related to cAMP turnover. By focusing on new molecular connections of protein kinase A (PKA) we hope to understand how distinct PKA subunits along with activation of beta adrenergic receptors have opposing effects on cell proliferation. So far the results that we have obtained emphasize possible crosstalk between PKA subunits and different GTPases. Some of the identified protein:protein interactions are formed in a cAMP- dependent manner. Overall we suggest that PKA subunits have a prospective role in coordinating GTPase function. We propose that novel complexes of PKA:GTPases are involved in transmitting and enhancing proliferative signals triggered by GPCRs. Reconstruction and analyses of these dynamic signaling events involving GTPases will help us to understand the opposing functions of PKA in cell growth and differentiation. By doing so we hope to resolve some puzzles concerning how and when connections between pathways are established and how information flows through signaling crossroads that distinct subunits of PKA appear to represent.

The specificity of receptor initiated signaling responses is encoded by spatial and temporal dynamics of downstream signaling networks. These networks, initiating from e.g. the G protein-coupled receptor (GPCR) superfamily and receptor tyrosine kinases (RTK), tightly regulate signaling pathways at several critical points via feedback loops and crosstalk among other pathways. Despite the fact that signaling networks are complex, signaling crosstalk can often be explained by simple and occasionally surprising molecular interactions. Elucidation of novel physical connection between critically controlled receptor pathways and central molecular switches such as kinases and GTPase was the primary goal of the FWF project P22608. We characterized several novel functional connections between the prototypical protein kinase A (PKA) and two small GTPases, Rac1 and G?i respectively. Our published study on the formation of the PKA:Rac complex offers an additional explanation how cAMP-fluxes might contribute to deregulated cell growth in a cell dependent manner. The presented mechanism is an example how components of plasma membrane receptor pathways like RTKs and GPCRs dynamically interact to control and adjust signal transmission into the nucleus. We also characterized the complex between PKA and G?i. It is actually the discovery of an unexpected novel function of PKA regulatory subunits: Following cAMP-binding to PKA regulatory subunits a complex with G?i is formed. This dynamic interaction changes the sensitivity and increases the amplitude of Gai-receptor mediated signal transmission in human cells. The exact molecular mechanism was found in bakers yeast (S. cerevisiae) as well. Our published data indicate that this protein:protein interaction (PPI) has been evolutionary conserved for at least 1,5 billion years. The formation of this cAMP initiated protein complex represents a novel mechanism how cells respond to changes of the environment. We demonstrated that hormone-regulated signaling cascades are interconnected and they participate in processes of cellular adaption. Besides publication of this mechanism we filed an EU patent and we submitted a patent application focusing on the perturbation of this novel cross-talk. In addition we further developed the cell-based protein-fragment complementation assay (PCA) technology to identify, characterize, and chemically perturb critical protein complexes. We showed that besides studying dynamics of PPI, our cell based reporter can be used in model organism such as bakers yeast, zebra fish embryos and the living mouse for tracking PPIs. We just finalized our PKA network study which describes a surprising new binary interaction between GPCRs and the PKA. It is the identification of the first GPCR with intrinsic scaffolding function for PKA.