Ubiquitylation of BBSome is required for ciliary assembly and signaling.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, renal abnormalities, postaxial polydactyly, and developmental defects. Genes mutated in BBS encode for components and regulators of the BBSome, an octameric complex that controls the trafficking of cargos and receptors within the primary cilium. Although both structure and function of the BBSome have been extensively studied, the impact of ubiquitin signaling on BBSome is largely unknown. We identify the E3 ubiquitin ligase PJA2 as a novel resident of the ciliary compartment and regulator of the BBSome. Upon GPCR-cAMP stimulation, PJA2 ubiquitylates BBSome subunits. We demonstrate that ubiquitylation of BBS1 at lysine 143 increases the stability of the BBSome and promotes its binding to BBS3, an Arf-like GTPase protein controlling the targeting of the BBSome to the ciliary membrane. Downregulation of PJA2 or expression of a ubiquitylation-defective BBS1 mutant (BBS1K143R ) affects the trafficking of G-protein-coupled receptors (GPCRs) and Shh-dependent gene transcription. Expression of BBS1K143R in vivo impairs cilium formation, embryonic development, and photoreceptors' morphogenesis, thus recapitulating the BBS phenotype in the medaka fish model.

Targeted inhibition of ubiquitin signaling reverses metabolic reprogramming and suppresses glioblastoma growth.

Glioblastoma multiforme (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population; its high recurrence rate and resistance to current therapeutics urgently demand a better therapy. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. UPS deregulation is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, the UPS represents a potentially valuable target for GBM treatment. Using an integrated approach that includes proteomics, transcriptomics and metabolic profiling, we identify praja2, a RING E3 ubiquitin ligase, as the key component of a signaling network that regulates GBM cell growth and metabolism. Praja2 is preferentially expressed in primary GBM lesions expressing the wild-type isocitrate dehydrogenase 1 gene (IDH1). Mechanistically, we found that praja2 ubiquitylates and degrades the kinase suppressor of Ras 2 (KSR2). As a consequence, praja2 restrains the activity of downstream AMP-dependent protein kinase in GBM cells and attenuates the oxidative metabolism. Delivery in the brain of siRNA targeting praja2 by transferrin-targeted self-assembling nanoparticles (SANPs) prevented KSR2 degradation and inhibited GBM growth, reducing the size of the tumor and prolonging the survival rate of treated mice. These data identify praja2 as an essential regulator of cancer cell metabolism, and as a potential therapeutic target to suppress GBM growth.

Pathophysiology of Primary Cilia: Signaling and Proteostasis Regulation.

Primary cilia are microtubule-based, non-motile sensory organelles present in most types of growth-arrested eukaryotic cells. They are transduction hubs that receive and transmit external signals to the cells in order to control growth, differentiation and development. Mutations of genes involved in the formation, maintenance or disassembly of ciliary structures cause a wide array of developmental genetic disorders, also known as ciliopathies. The primary cilium is formed during G1 in the cell cycle and disassembles at the G2/M transition. Following the completion of the cell division, the cilium reassembles in G1. This cycle is finely regulated at multiple levels. The ubiquitin-proteasome system (UPS) and the autophagy machinery, two main protein degradative systems in cells, play a fundamental role in cilium dynamics. Evidence indicate that UPS, autophagy and signaling pathways may act in synergy to control the ciliary homeostasis. However, the mechanisms involved and the links between these regulatory systems and cilium biogenesis, dynamics and signaling are not well defined yet. Here, we discuss the reciprocal regulation of signaling pathways and proteolytic machineries in the control of the assembly and disassembly of the primary cilium, and the impact of the derangement of these regulatory networks in human ciliopathies.

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation.

The primary cilium is a microtubule-based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin-proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G-protein-coupled receptor (GPCR)-cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2-UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.

Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system.

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.

The role of compartmentalized signaling pathways in the control of mitochondrial activities in cancer cells.

Mitochondria are the powerhouse organelles present in all eukaryotic cells. They play a fundamental role in cell respiration, survival and metabolism. Stimulation of G-protein coupled receptors (GPCRs) by dedicated ligands and consequent activation of the cAMP·PKA pathway finely couple energy production and metabolism to cell growth and survival. Compartmentalization of PKA signaling at mitochondria by A-Kinase Anchor Proteins (AKAPs) ensures efficient transduction of signals generated at the cell membrane to the organelles, controlling important aspects of mitochondrial biology. Emerging evidence implicates mitochondria as essential bioenergetic elements of cancer cells that promote and support tumor growth and metastasis. In this context, mitochondria provide the building blocks for cellular organelles, cytoskeleton and membranes, and supply all the metabolic needs for the expansion and dissemination of actively replicating cancer cells. Functional interference with mitochondrial activity deeply impacts on cancer cell survival and proliferation. Therefore, mitochondria represent valuable targets of novel therapeutic approaches for the treatment of cancer patients. Understanding the biology of mitochondria, uncovering the molecular mechanisms regulating mitochondrial activity andmapping the relevant metabolic and signaling networks operating in cancer cells will undoubtly contribute to create a molecular platform to be used for the treatment of proliferative disorders. Here, we will highlight the emerging roles of signaling pathways acting downstream to GPCRs and their intersection with the ubiquitin proteasome system in the control of mitochondrial activity in different aspects of cancer cell biology.

Counterregulation of cAMP-directed kinase activities controls ciliogenesis.

The primary cilium emanates from the cell surface of growth-arrested cells and plays a central role in vertebrate development and tissue homeostasis. The mechanisms that control ciliogenesis have been extensively explored. However, the intersection between GPCR signaling and the ubiquitin pathway in the control of cilium stability are unknown. Here we observe that cAMP elevation promotes cilia resorption. At centriolar satellites, we identify a multimeric complex nucleated by PCM1 that includes two kinases, NEK10 and PKA, and the E3 ubiquitin ligase CHIP. We show that NEK10 is essential for ciliogenesis in mammals and for the development of medaka fish. PKA phosphorylation primes NEK10 for CHIP-mediated ubiquitination and proteolysis resulting in cilia resorption. Disarrangement of this control mechanism occurs in proliferative and genetic disorders. These findings unveil a pericentriolar kinase signalosome that efficiently links the cAMP cascade with the ubiquitin-proteasome system, thereby controlling essential aspects of ciliogenesis.

Mitochondrial AKAP1 supports mTOR pathway and tumor growth.

Mitochondria are the powerhouses of energy production and the sites where metabolic pathway and survival signals integrate and focus, promoting adaptive responses to hormone stimulation and nutrient availability. Increasing evidence suggests that mitochondrial bioenergetics, metabolism and signaling are linked to tumorigenesis. AKAP1 scaffolding protein integrates cAMP and src signaling on mitochondria, regulating organelle biogenesis, oxidative metabolism and cell survival. Here, we provide evidence that AKAP1 is a transcriptional target of Myc and supports the growth of cancer cells. We identify Sestrin2, a leucine sensor and inhibitor of the mammalian target of rapamycin (mTOR), as a novel component of the complex assembled by AKAP1 on mitochondria. Downregulation of AKAP1 impaired mTOR pathway and inhibited glioblastoma growth. Both effects were reversed by concomitant depletion of AKAP1 and sestrin2. High levels of AKAP1 were found in a wide variety of high-grade cancer tissues. In lung cancer, AKAP1 expression correlates with high levels of Myc, mTOR phosphorylation and reduced patient survival. Collectively, these data disclose a previously unrecognized role of AKAP1 in mTOR pathway regulation and cancer growth. AKAP1/mTOR signal integration on mitochondria may provide a new target for cancer therapy.

Proteolytic control of neurite outgrowth inhibitor NOGO-A by the cAMP/PKA pathway.

Protein kinase A (PKA) controls major aspects of neurite outgrowth and morphogenesis and plays an essential role in synaptic plasticity and memory. However, the molecular mechanism(s) of PKA action on neurite sprouting and activity are still unknown. Here, we report that in response to neurotrophin or cAMP stimulation the RING ligase praja2 ubiquitinates and degrades NOGO-A, a major inhibitor of neurite outgrowth in mammalian brain. Genetic silencing of praja2 severely inhibited neurite extension of differentiating neuroblastoma cells and mesencephalic neurons and axon outgrowth and sprouting of striatal terminals in developing rat brain. This phenotype was rescued when both praja2 and NOGO-A were depleted, suggesting that NOGO-A is, indeed, a biologically relevant target of praja2 in neuronal cells. Our findings unveil a novel mechanism that functionally couples cAMP signaling with the proteolytic turnover of NOGO-A, positively impacting on neurite outgrowth in mammalian brain.

Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates Hippo signalling and supports glioblastoma growth.

Human glioblastoma is the most frequent and aggressive form of brain tumour in the adult population. Proteolytic turnover of tumour suppressors by the ubiquitin-proteasome system is a mechanism that tumour cells can adopt to sustain their growth and invasiveness. However, the identity of ubiquitin-proteasome targets and regulators in glioblastoma are still unknown. Here we report that the RING ligase praja2 ubiquitylates and degrades Mob, a core component of NDR/LATS kinase and a positive regulator of the tumour-suppressor Hippo cascade. Degradation of Mob through the ubiquitin-proteasome system attenuates the Hippo cascade and sustains glioblastoma growth in vivo. Accordingly, accumulation of praja2 during the transition from low- to high-grade glioma is associated with significant downregulation of the Hippo pathway. These findings identify praja2 as a novel upstream regulator of the Hippo cascade, linking the ubiquitin proteasome system to deregulated glioblastoma growth.