AKAP150 knockout- and mutant knock-in alleles reveal an unexpected role of the adaptor in anchoring phosphatase 2B for efficient insulin secretion from pancreatic β-cells and thus glucose homeostasis.

α 1D -Adrenergic receptors (ARs) are key regulators of cardiovascular system function that increase blood pressure and promote vascular remodeling. Unfortunately, little information exists about the signaling pathways used by this important G protein-coupled receptor (GPCR). We recently discovered that α 1D -ARs form a “signalosome” with multiple members of the dystrophin-associated protein complex (DAPC) to become functionally expressed at the plasma membrane and bind ligands. However, the molecular mechanism by which the DAPC imparts functionality to the α 1D -AR signalosome remains a mystery. To test the hypothesis that previously unidentified molecules are recruited to the α 1D -AR signalosome, we performed an extensive proteomic analysis on each member of the DAPC. Bioinformatic analysis of our proteomic data sets detected a common interacting protein of relatively unknown function, α-catulin. Coimmunoprecipitation and blot overlay assays indicate that α-catulin is directly recruited to the α 1D -AR signalosome by the C-terminal domain of α-dystrobrevin-1 and not the closely related splice variant α-dystrobrevin-2. Proteomic and biochemical analysis revealed that α-catulin supersensitizes α 1D -AR functional responses by recruiting effector molecules to the signalosome. Taken together, our study implicates α-catulin as a unique regulator of GPCR signaling and represents a unique expansion of the intricate and continually evolving array of GPCR signaling networks.

Background: AKAP220 organizes the signaling enzymes PKA, GSK-3, and phosphoprotein phosphatase PP1.Results: AKAP220 interacts with the scaffolding protein IQGAP1 to assimilate and process calcium and cAMP signals at leading edges of migrating cells.Conclusion: AKAP220/IQGAP1 networks position calcium and cAMP-responsive signaling enzymes near substrates at the +TIPs of growing microtubules.Significance: Anchored kinase/microtubule effector protein networks propagate cell motility.

Protein kinase A-anchoring proteins (AKAPs) influence fundamental cellular processes by directing the cAMP-dependent protein kinase (PKA) toward its intended substrates. In this report we describe the identification and characterization of a ternary complex of AKAP220, the PKA holoenzyme, and the IQ domain GTPase-activating protein 2 isoform (IQGAP2) that is enriched at cortical regions of the cell. Formation of an IQGAP2-AKAP220 core complex initiates a subsequent phase of protein recruitment that includes the small GTPase Rac. Biochemical and molecular biology approaches reveal that PKA phosphorylation of Thr-716 on IQGAP2 enhances association with the active form of the Rac GTPase. Cell-based experiments indicate that overexpression of an IQGAP2 phosphomimetic mutant (IQGAP2 T716D) enhances the formation of actin-rich membrane ruffles at the periphery of HEK 293 cells. In contrast, expression of a nonphosphorylatable IQGAP2 T716A mutant or gene silencing of AKAP220 suppresses formation of membrane ruffles. These findings imply that IQGAP2 and AKAP220 act synergistically to sustain PKA-mediated recruitment of effectors such as Rac GTPases that impact the actin cytoskeleton.

Cell proliferation involves the coordinated expression of protein encoding genes that control progression through the cell cycle. These regulators include cyclin D1, a growth factor sensor that integrates extracellular signals with the core cell cycle machinery (35). Overexpression of cyclin D1, which accelerates entry into S phase, is frequently found in human cancers and is often associated with a poor prognosis (34). Common mechanisms for cyclin D1 overexpression in cancer cells are gene amplification and gene rearrangements, causing abnormally elevated levels of transcript and protein. Such genomic aberrations are not a feature of all cancer cells that overexpress cyclin D1, suggesting the involvement of alternative transcriptional upregulation mechanisms.In eukaryotes, expression of protein-encoding genes is carried out by the RNA polymerase II (Pol II)-dependent transcription machinery. Initiation of transcription is mediated by members of either the TFIID or the SAGA (TFTC, PCAF, SAGA) family of coactivator complexes (8,12,27,30,38,40). TFIID complexes contain the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) (8, 38). The SAGA family of complexes does not contain TBP and instead is composed of the histone acetyltransferase Gcn5 and a subset of TAFs present in TFIID. Members of the SAGA family are essential for transcription of only 10% of yeast genes, which suggests that TFIID complexes are responsible for the majority of RNA Pol II-dependent transcription (17). Within TFIID, TBP and the ϳ14 TAFs interact to form a trilobed structure, as determined by immunoelectron microscopy (20). TAF1, the largest subunit of TFIID, makes extensive contacts with TBP and many other TAFs, including TAF7.Human TAF1 is a unique molecule in that it possesses intrinsic protein kinase, histone acetyltransferase (HAT), and ubiquitinactivating and -conjugating activities (6,24,29). We previously reported that TAF1 HAT activity is required for efficient transcription of cyclin D1 and cyclin A genes in mammalian cells (7,16). The HAT domain of TAF1 is located in the central region of the protein and is highly conserved in all eukaryotes. In vitro, histones H3 and H4 and the basal transcription factors TFIIE␤ and TFIIF have been identified as the substrates for TAF1 HAT activity (18,24). Recent work has suggested that TAF7, another TFIID subunit, may play a pivotal role in the regulation of TAF1 HAT activity. Gegonne et al. identified TAF7 as a protein capable of directly binding to TAF1 and inhibiting TAF1 HAT activity (10). TAF1 also contains two independent serine/threonine kinase domains, one in the N terminus (NTK) and one in the C terminus (CTK) of the protein. Kinase activity has been demonstrated in vitro for human, yeast, and Drosophila TAF1 (6,22,23). Both domains are classified as atypical kinases and share little amino acid homology with each other; however, the NTK and CTK domains both are capable of autophosphorylation and transphosphorylation of substrates such as the TFIID subunit TAF7 (11).I...

Scaffolding the calcium/calmodulin-dependent phosphatase 2B (PP2B, calcineurin) focuses and insulates termination of local second messenger responses. Conformational flexibility in regions of intrinsic disorder within A-kinase anchoring protein 79 (AKAP79) delineates PP2B access to phosphoproteins. Structural analysis by negative-stain electron microscopy (EM) reveals an ensemble of dormant AKAP79-PP2B configurations varying in particle length from 160 to 240 Å. A short-linear interaction motif between residues 337–343 of AKAP79 is the sole PP2B-anchoring determinant sustaining these diverse topologies. Activation with Ca2+/calmodulin engages additional interactive surfaces and condenses these conformational variants into a uniform population with mean length 178 ± 17 Å. This includes a Leu-Lys-Ile-Pro sequence (residues 125–128 of AKAP79) that occupies a binding pocket on PP2B utilized by the immunosuppressive drug cyclosporin. Live-cell imaging with fluorescent activity-sensors infers that this region fine-tunes calcium responsiveness and drug sensitivity of the anchored phosphatase.

Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an "actin barrier" that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis. yet only approximately 1.5 L of urine is excreted. The majority of this water is reabsorbed from the luminal fluid of the nephron (1). Regulated water reabsorption in response to dehydration occurs through aquaporin-2 (AQP2) water pores in the principal cells of the collecting duct (2). This is stimulated by the hormone arginine vasopressin (AVP). Vasopressin induces PKA phosphorylation of serine 256 on AQP2 and stimulates its translocation from intracellular vesicles to the apical membranes of cells lining the collecting ducts. Reabsorption of water through the kidney preserves fluid balance and results in more concentrated urine (3, 4). Conversely, when an animal ingests excess water, decreased plasma osmolality inhibits vasopressin release, and AQP2 is recovered from the apical membrane by endocytosis. This renders the collecting duct impermeable to water, thereby diverting excess water through the ureter to the bladder.Not surprisingly, defects in AQP2 trafficking have pathophysiological outcomes. For example, nephrogenic diabetes insipidus (NDI) is associated with impaired vasopressin signaling to AQP2 (5-8). Symptoms include excessive thirst, excretion of a large volume of dilute urine, and electrolyte imbalances, including hypernatremia (1, 5). Hereditary forms of this disease appear in patients with inactivating mutations in the V2 vasopressin receptor (V2R) or AQP2 (9-12). Thus, understanding the molecular mechanisms that govern bidirectional control of AQP2 location may lead to new therapeutic approaches for the treatment of NDI.Although the enzymes and effector proteins that govern AQP2 in...

Background: AKAPs integrate intracellular signals by sequestering PKA with other kinases. Results: Phosphorylation of Thr-1132 on AKAP220 initiates GSK3␤ recruitment, and PKA activity drives the release of GSK3␤ from the complex. Conclusion: Cross-talk between PKA and GSK3␤ is optimized in the context of AKAP220 multienzyme complexes. Significance: Signal responsive assembly of enzyme complexes may represent a general mechanism to diversify transduction through AKAPs.