Neurochondrin is an atypical RIIα-specific A-kinase anchoring protein

Protein kinase activity is regulated not only by direct strategies affecting activity but also by spatial and temporal regulatory mechanisms. Kinase signaling pathways are coordinated by scaffolding proteins that orchestrate the assembly of multi-protein complexes. One family of such scaffolding proteins are the A-kinase anchoring proteins (AKAPs). AKAPs share the commonality of binding cAMP-dependent protein kinase (PKA). In addition, they bind further signaling proteins and kinase substrates and tether such multi-protein complexes to subcellular locations. The A-kinase binding (AKB) domain of AKAPs typically contains a conserved helical motif that interacts directly with the dimerization/docking (D/D) domain of the regulatory subunits of PKA. Based on a pull-down proteomics approach, we identified neurochondrin (neurite-outgrowth promoting protein) as a previously unidentified AKAP. Here, we show that neurochondrin interacts directly with PKA through a novel mechanism that involves two distinct binding regions. In addition, we demonstrate that neurochondrin has strong isoform selectivity towards the RII{alpha} subunit of PKA with nanomolar affinity. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases

AKAP18 PKA RII alpha structure reveals crucial anchor points for recognition of regulatory subunits of PKA

A-kinase anchoring proteins (AKAPs) interact with the dimerization/docking (D/D) domains of regulatory subunits of the ubiquitous protein kinase A (PKA). AKAPs tether PKA to defined cellular compartments establishing distinct pools to increase the specificity of PKA signalling. Here, we elucidated the structure of an extended PKA-binding domain of AKAP18{beta} bound to the D/D domain of the regulatory RII{alpha} subunits of PKA. We identified three hydrophilic anchor points in AKAP18{beta} outside the core PKA-binding domain, which mediate contacts with the D/D domain. Such anchor points are conserved within AKAPs that bind regulatory RII subunits of PKA. We derived a different set of anchor points in AKAPs binding regulatory RI subunits of PKA. In vitro and cell-based experiments confirm the relevance of these sites for the interaction of RII subunits with AKAP18 and of RI subunits with the RI-specific smAKAP. Thus we report a novel mechanism governing interactions of AKAPs with PKA. The sequence specificity of each AKAP around the anchor points and the requirement of these points for the tight binding of PKA allow the development of selective inhibitors to unequivocally ascribe cellular functions to the AKAP18-PKA and other AKAP-PKA interactions

Identification of a novel A-kinase anchoring protein 18 isoform and evidence for its role in the vasopressin-induced aquaporin-2 shuttle in renal principal cells

Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18delta, was identified. AKAP18delta functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18delta was identified on the same intracellular vesicles as AQP2 and PKA. AVP not only recruited AQP2, but also AKAP18delta to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18delta and PKA. The data suggest that AKAP18delta is involved in the AQP2 shuttle

Mutant phosphodiesterase 3A protects from hypertension-induced cardiac damage

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca(2+) imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The β-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca(2+) cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage