GPR61 anchoring of PKA consolidates GPCR and cAMP signaling

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions

Functional analyses of compartmentalized binary Myc interactions

c-Myc is a member of the bHLH-LZ protein family which needs to heterodimerize with Max to perform its critical functions as a transcription factor. Thereby it regulates growth, cell proliferation, DNA replication, cell cycle progression, differentiation, and metabolism. Deregulation of Myc expression contributes to the etiology and progression of cancer. Due to its huge impact on cellular processes, deregulated Myc signaling occurs in almost all types of human cancer. In addition to c-Myc, the variants v-Myc, N-Myc, L-Myc have been studied intensively. In this work, differences of the oncogenic potentials of the Myc variants were analyzed using transformation assays based on avian cell culture systems. v-Myc displayed the highest transforming potential followed by N-Myc, c-Myc, and L-Myc. The transformation potential correlates with the protein-protein interaction (PPI) pattern which was quantified using a protein fragment complementation assay (PCA). In addition to the studies of Myc:Max Renilla Luciferase (RLuc) PCA dynamics using cellular second messenger molecules, the subcellular localization using Venus YFP (Ven) PCA was determined. Besides nuclear localization of Myc332-439:Max and Max:Max, we showed for the first time that Ven PCA tagged full length Myc:Max complexes are restricted to the nucleus of quail embryonic fibroblasts. Finally, a new PPI was analyzed which might link BASP1 functions to Myc signaling. The Myc target gene BASP1 is known to inhibit Myc-induced cell transformation. Calmodulin (CaM) interacts with BASP1 and could be functionally connected to BASP1-mediated Myc inhibition. We have characterized the binding interface of the Myc:CaM PPI, which involves the basic region of Myc. We believe that targeting known and new PPIs of Myc is a promising strategy to reduce Myc variant driven cell proliferation.submitted by Ruth RöckInnsbruck, Univ., Master-Arb., 2015(VLID)43974

Kidney development - recent insights from technological advances

The kidney is a complex organ, and how it forms is a fascinating process. New technologies, such as single-cell transcriptomics, and enhanced imaging modalities are offering new approaches to understand the complex and intertwined processes during embryonic kidney development