Perspectives on: Cyclic Nucleotide Microdomains and Signaling Specificity

The author provides an introduction to a special issue on cyclic nucleotides concerning four areas in cAMP compartmentation

A Flagellar A-Kinase Anchoring Protein with Two Amphipathic Helices Forms a Structural Scaffold in the Radial Spoke Complex

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa and Dpy-30 domains, in four non-PKA spoke proteins involved in the assembly and modulation of the complex. Interestingly, one AH can bind both RIIa and Dpy-30 domains in vitro. Thus, AHs and D/D domains constitute a versatile yet potentially promiscuous system for localizing various effector mechanisms. These results greatly expand the current concept about anchoring mechanisms and AKAPs

Identification of a Novel TGFβ/PKA Signaling Transduceome in Mediating Control of Cell Survival and Metastasis in Colon Cancer

Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3′ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing

Optic atrophy 1 is an A-kinase anchoring protein on lipid droplets that mediates adrenergic control of lipolysis

International audienceAdrenergic stimulation of adipocytes yields a cAMP signal that activates protein kinase A (PKA). PKA phosphory-lates perilipin, a protein localized on the surface of lipid droplets that serves as a gatekeeper to regulate access of lipases converting stored triglycerides to free fatty acids and glycerol in a phosphorylation-dependent manner. Here, we report a new function for optic atrophy 1 (OPA1), a protein known to regulate mitochondrial dynamics, as a dual-specificity A-kinase anchoring protein associated with lipid droplets. By a variety of protein interaction assays, immunoprecipitation and immunolo-calization experiments, we show that OPA1 organizes a supramolecular complex containing both PKA and perili-pin. Furthermore, by a combination of siRNA-mediated knockdown, reconstitution experiments using full-length OPA1 with or without the ability to bind PKA or truncated OPA1 fused to a lipid droplet targeting domain and cellular delivery of PKA anchoring disruptor peptides, we demonstrate that OPA1 targeting of PKA to lipid droplets is necessary for hormonal control of perilipin phosphoryla-tion and lipolysis

Rapsyn mediates subsynaptic anchoring of PKA type I and stabilisation of acetylcholine receptor in vivo

The stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12 and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction. Molecular modelling, immunoprecipitation and bimolecular fluorescence complementation approaches identify an α-helical stretch of rapsyn to be crucial for binding to the dimerisation and docking domain of PKA type I. When expressed in live mouse muscle, a peptide encompassing the rapsyn α-helical sequence efficiently delocalises PKA type I from the neuromuscular junction. The same peptide, as well as a rapsyn construct lacking the α-helical domain, induces severe alteration of acetylcholine receptor turnover as well as fragmentation of synapses. This shows that rapsyn anchors PKA type I in close proximity to the postsynaptic membrane and suggests that this function is essential for synapse maintenance