Control of PKA stability and signalling by RING ligase praja2

Ligand-mediated activation of G-Protein Coupled Receptors (GPCRs) mobilises compartmentalised pulses of second messengers like cAMP. The main cellular effector of cAMP is Protein Kinase A (PKA) which is assembled as inactive holoenzyme consisting of two regulatory (R) and two catalytic (PKAc) subunits. cAMP binding to the R subunits dissociates the holoenzyme and releases the catalytic moiety, which phosphorylates a wide array of cellular proteins1. Re-association of the PKAc and R components terminates the signal. The rate of association and dissociation of R and PKAc subunits determines the intensity and the duration of the kinase activity, influencing complex biological phenotypes such as long term memory, differentiation and apoptosis2. Proteolysis of R subunits has been proposed as mechanism to sustain signalling downstream of PKAc3, although no enzyme targeting R subunits had been identified. Here we report that praja2, a RING E3 ligase widely expressed in mammalian cells, controls the stability of R subunits. Praja2 forms a stable complex with and is phosphorylated by PKA. Elevated cAMP levels promote Praja2-mediated ubiquitination and subsequent proteolysis of compartmentalised R subunits. Functional experiments in neuroblastoma cells and rat brains show that praja2 is required for efficient nuclear cAMP signalling and for PKA-mediated long-term potentiation (LTP). These findings indicate that praja2 regulates the total concentration of R subunits, thereby tuning the strength and duration of PKA signal output in response to the cAMP concentration

152 Insights into the pathogenesis of HHV8-driven body cavity-based lymphoma

Human herpesvirus 8 is associated with the development of primary effusion lymphoma (PEL), an aggressive non-Hodgkin’s lymphoma characterized by the proliferation of the malignant lymphocytes almost exclusively in large serous cavities. The mechanisms involved in the preferential tropism for serous cavities and in the aggressive course of PEL remain to be fully clarified. To study the role of host microenvironment in PEL progression, we previously compared the antineoplastic activity of a murine interferon◊-expressing lentiviral vector (mIFN-◊-LV) to that of a human IFN-◊-LV in a murine model of peritoneal PEL. We demonstrated that in vivo targeting of the murine microenvironment showed an antineoplastic activity comparable to that observed with the hIFN-◊-LV. These findings highlighted the relevant role of body cavity environment in PEL growth and indicated that modulation of microenvironment may impair PEL growth in vivo. By using cocultures of PEL cell lines with human mesothelial cells (HMC), we mimicked the interactions existing in body cavities to analyze the mechanisms involved in PEL progression. PEL cells induced a myofibroblastic morphology in HMC, paralleled by an expression profile indicative of the occurrence of epithelial-mesenchymal transition (EMT). Moreover, HMC increased proliferation and resistance to apoptosis of PEL cells. These data indicate that PEL cells induce EMT in HMC and fibrosis of serous membranes. In turn, HMC modulate PEL cell turnover, thus providing a milieu favorable to PEL progression. These findings open new perspectives into the mechanisms involved in PEL progression and may indicate new targets for PEL treatment

Expression of the Ring Ligase PRAJA2 in Thyroid Cancer

Introduction: In thyroid cells, binding of TSH to its receptor increases cAMP levels, sustaining thyrocytes growth and hormone production. The main cAMP effector enzyme is protein kinase A (PKA). Praja2 is a widely expressed RING (Really Interesting New Gene) ligase, which degrades the regulatory subunits of PKA, thus controlling the strengthandduration ofPKAsignaling in response to cAMP. Differentiated thyroid cancer expresses a functional TSH receptor, and its growth and progression are positively regulated by TSH and cAMP signaling. Aim: We aimed to analyze the expression of praja2 in a group of 36 papillary thyroid cancer (PTC), 14 benign nodules, and six anaplastic thyroid cancers (ATC). Methods: We measured praja2 mRNA levels by quantitative RT-PCR and praja2 expression by Western blot and immunohistochemistry. Possible association between praja2 mRNA and the presence of known mutations was evaluated. Results:We foundastatistical significant increase ofmRNAlevels inPTCtissue samples,comparedwith benign nodules and ATC. In particular, mRNA levels were maximal in differentiated thyroid cancer (PTC), progressively decreasing in more aggressive tumors, ATC having the lowest amount of praja2 mRNA. Accordingly, higher levels of praja2 protein were detected in lysates from PTC, compared with ATC. By immunohistochemistry, in PTC sections we observed a marked increase of cytoplasmic praja2 signal, which significantly decreased in less differentiated thyroid tumors, completely disappearing in ATC. Studies in cultured cells stably expressing RET/PTC1 oncogene or mutant BRAF revealed a direct correlationbetweenpraja2mRNAlevelsandmalignant phenotype of transformed cells. Similar results were obtained using thyroid cancer tissues carrying the same mutations. Conclusions: praja2 is markedly overexpressed in differentiated thyroid cancer, and its levels inversely correlate with the malignant phenotype of the tumor. Thus, praja2 is a novel cancer-related gene whose expression is linked to the histotype and mutational status of the thyroid tumor

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 (BBS1 K143R) affects the trafficking of G-proteincoupled receptors (GPCRs) and Shh-dependent gene transcription. Expression of BBS1 K143R 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