Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Mutations causing acrodysostosis-2 facilitate activation of phosphodiesterase 4D3.

Type 2 acrodysostosis (ACRDYS2), a rare developmental skeletal dysplasia characterized by short stature, severe brachydactyly and facial dysostosis, is caused by mutations in the phosphodiesterase (PDE) 4D (PDE4D) gene. Several arguments suggest that the mutations should result in inappropriately increased PDE4D activity, however, no direct evidence supporting this hypothesis has been presented, and the functional consequences of the mutations remain unclear. We evaluated the impact of four different PDE4D mutations causing ACRDYS2 located in different functional domains on the activity of PDE4D3 expressed in Chinese hamster ovary cells. Three independent approaches were used: the direct measurement of PDE activity in cell lysates, the evaluation of intracellular cAMP levels using an EPAC-based (exchange factor directly activated by cAMP) bioluminescence resonance energy transfer sensor , and the assessment of PDE4D3 activation based on electrophoretic mobility. Our findings indicate that PDE4D3s carrying the ACRDYS2 mutations are more easily activated by protein kinase A-induced phosphorylation than WT PDE4D3. This occurs over a wide range of intracellular cAMP concentrations, including basal conditions, and result in increased hydrolytic activity. Our results provide new information concerning the mechanism whereby the mutations identified in the ACRDYS2 dysregulate PDE4D activity, and give insights into rare diseases involving the cAMP signaling pathway. These findings may offer new perspectives into the selection of specific PDE inhibitors and possible therapeutic intervention for these patients.

Knock-In of the Recurrent R368X Mutation of PRKAR1A that Represses cAMP-Dependent Protein Kinase A Activation: A Model of Type 1 Acrodysostosis.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (µCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.

Engineered stabilization and structural analysis of the autoinhibited conformation of PDE4.

Phosphodiesterase 4 (PDE4) is an essential contributor to intracellular signaling and an important drug target. The four members of this enzyme family (PDE4A to -D) are functional dimers in which each subunit contains two upstream conserved regions (UCR), UCR1 and -2, which precede the C-terminal catalytic domain. Alternative promoters, transcriptional start sites, and mRNA splicing lead to the existence of over 25 variants of PDE4, broadly classified as long, short, and supershort forms. We report the X-ray crystal structure of long form PDE4B containing UCR1, UCR2, and the catalytic domain, crystallized as a dimer in which a disulfide bond cross-links cysteines engineered into UCR2 and the catalytic domain. Biochemical and mass spectrometric analyses showed that the UCR2-catalytic domain interaction occurs in trans, and established that this interaction regulates the catalytic activity of PDE4. By elucidating the key structural determinants of dimerization, we show that only long forms of PDE4 can be regulated by this mechanism. The results also provide a structural basis for the long-standing observation of high- and low-affinity binding sites for the prototypic inhibitor rolipram.

Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.

Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction of other PDE4 isoforms that can be expected to be targeted to different signaling complexes and exert distinct effects on compartmentalized cAMP signaling.

Early initiation of enzyme replacement therapy for the mucopolysaccharidoses.

The mucopolysaccharidoses (MPS), a group of rare genetic disorders caused by defects in glycosaminoglycan (GAG) catabolism, are progressive, multi-systemic diseases with a high burden of morbidity. Enzyme replacement therapy (ERT) is available for MPS I, II, and VI, and may improve walking ability, endurance, and pulmonary function as evidenced by data from pivotal trials and extension studies. Despite these demonstrable benefits, cardiac valve disease, joint disease, and skeletal disease, all of which cause significant morbidity, do not generally improve with ERT if pathological changes are already established. Airway disease improves, but usually does not normalize. These limitations can be well understood by considering the varied functions of GAG in the body. Disruption of GAG catabolism has far-reaching effects due to the triggering of secondary pathogenic cascades. It appears that many of the consequences of these secondary pathogenic events, while they may improve on treatment, cannot be fully corrected even with long-term exposure to enzyme, thereby supporting the treatment of patients with MPS before the onset of clinical disease. This review examines the data from clinical trials and other studies in human patients to explore the limits of ERT as currently used, then discusses the pathophysiology, fetal tissue studies, animal studies, and sibling reports to explore the question of how early to treat an MPS patient with a firm diagnosis. The review is followed by an expert opinion on the rationale for and the benefits of early treatment.

Congenital joint dislocations caused by carbohydrate sulfotransferase 3 deficiency in recessive Larsen syndrome and humero-spinal dysostosis.

Deficiency of carbohydrate sulfotransferase 3 (CHST3; also known as chondroitin-6-sulfotransferase) has been reported in a single kindred so far and in association with a phenotype of severe chondrodysplasia with progressive spinal involvement. We report eight CHST3 mutations in six unrelated individuals who presented at birth with congenital joint dislocations. These patients had been given a diagnosis of either Larsen syndrome (three individuals) or humero-spinal dysostosis (three individuals), and their clinical features included congenital dislocation of the knees, elbow joint dysplasia with subluxation and limited extension, hip dysplasia or dislocation, clubfoot, short stature, and kyphoscoliosis developing in late childhood. Analysis of chondroitin sulfate proteoglycans in dermal fibroblasts showed markedly decreased 6-O-sulfation but enhanced 4-O-sulfation, confirming functional impairment of CHST3 and distinguishing them from diastrophic dysplasia sulphate transporter (DTDST)-deficient cells. These observations provide a molecular basis for recessive Larsen syndrome and indicate that recessive Larsen syndrome, humero-spinal dysostosis, and spondyloepiphyseal dysplasia Omani type form a phenotypic spectrum.

Serum hyaluronidase aberrations in metabolic and morphogenetic disorders.

Hyaluronidases are endo-glycosidases that degrade both hyaluronan (hyaluronic acid) (HA) and chondroitin sulfates. Deficiency of hyaluronidase activity has been predicted to result in a phenotype similar to that observed in mucopolysaccharidosis (MPS). In the present study, we surveyed a variety of patients with phenotypes similar to those observed in MPS, but without significant mucopolysacchariduria to determine if some are based on aberrations in serum hyaluronidase (Hyal-1) activity. The study included patients with well-characterized dysmorphic disorders occurring on genetic basis, as well as those of unkown etiology. The purpose of the study was to establish how wide spread were abnormalities in levels of circulating Hyal-1 activity. A simple and sensitive semi-quantitative zymographic procedure was used for the determination of activity. Levels of both beta-N-acetylglucosaminidase and beta-glucuronidase whose activities contribute to the total breakdown of hyaluronan (HA) were also measured, as well as the concentration of circulating HA. Among 48 patients with bone or connective tissue abnormalities, low levels of Hyal-1 activity were found in six patients compared to levels in 100 healthy donors (2.0-3.2 units/microL vs 6(+/- 1 SE) units/microL). These six patients exhibited a wide spectrum of clinical abnormalities, in particular shortened extremities: they included three patients with unknown causes of clinical symptoms, one patient with Sanfilippo disease, one of the seven patients with achondroplasia, and one with hypophosphotemic rickets. Normal levels of serum Hyal-1 activities were found in patients with Morquio disease, GM1 gangliosidosis, I cell-disease, 6 of the 7 patients with achondroplasia, Marfan's-syndrome and Ehlers-Danlos syndrome. No patient totally lacked serum Hyal-1 activity. Serum HA concentration was elevated in patients with Sanfilippo A and I-cell disease. Determination of serum and leukocyte Hyal-1 and serum HA may be useful to evaluate patients with metabolic and morphogenetic disorders.

Combined deficiency of beta-galactosidase and neuraminidase: natural history of the disease in the first 18 years of an American patient with late infantile onset form.

We describe the clinical findings over the first 18 years of a patient with a novel phenotype for galactosialidosis, the storage disease produced by the combined deficiency of beta-galactosidase and neuraminidase. Clinical findings in the first few months included somewhat unusual appearance and hepatosplenomegaly. Dysostosis multiplex was evident by age 2 1/2 years. Mitral and aortic valvular disease appeared over the next few years and cardiac disease has become the most important clinical problem. Foam cells were present in the bone marrow, and vacuolated lymphocytes were present in the peripheral blood smear. The patient had no neurological symptoms, cherry red spots, or intellectual deterioration during the first 18 years. Evidence presented elsewhere indicates that the basic defect in this late infantile form of galactosialidosis (as is thought to be true for the other forms of galactosialidosis) is a reduced amount of the 32 kDa phosphoglycoprotein which associates with beta-galactosidase and alpha-neuraminidase in lysosomes.