The CSN3 subunit of the COP9 signalosome interacts with the HD region of Sos1 regulating stability of this GEF protein

Sos1 is an universal, widely expressed Ras guanine nucleotide-exchange factor (RasGEF) in eukaryotic cells. Its N-terminal HD motif is known to be involved in allosteric regulation of Sos1 GEF activity through intramolecular interaction with the neighboring PH domain. Here, we searched for other cellular proteins also able to interact productively with the Sos1 HD domain. Using a yeast two-hybrid system, we identified the interaction between the Sos1 HD region and CSN3, the third component of the COP9 signalosome, a conserved, multi-subunit protein complex that functions in the ubiquitin-proteasome pathway to control degradation of many cellular proteins. The interaction of CSN3 with the HD of Sos1 was confirmed in vitro by GST pull-down assays using truncated mutants and reproduced in vivo by co-immunoprecipitation with the endogenous, full-length cellular Sos1 protein. In vitro kinase assays showed that PKD, a COP9 signalosome-associated-kinase, is able to phosphorylate Sos1. The intracellular levels of Sos1 protein were clearly diminished following CSN3 or PKD knockdown. A sizable fraction of the endogenous Sos1 protein was found ubiquitinated in different mammalian cell types. A significant reduction of RasGTP formation upon growth factor stimulation was also observed in CSN3-silenced as compared with control cells. Our data suggest that the interaction of Sos1 with the COP9 signalosome and PKD plays a significant role in maintenance of cellular Sos1 protein stability and homeostasis under physiological conditions and raises the possibility of considering the CSN/PKD complex as a potential target for design of novel therapeutic drugs.We thank R Brent for the pJG45-HeLa library and R. Jorge for help with yeast two-hybrid screening. J.M.R. received grant support from MINECO-FEDER (SAF2016-78852-R), ISCIII-MINECO (FIS-Intrasalud PI13/00703) and Spanish Association against Cancer (AECC). E.S. and A.F.M. were supported by grants from ISCIII-MINECO (FIS PI16/02137), JCyL (SA043U16-UIC 076) and Solorzano Foundation. E.S. and J.M.R. were also supported by ISCIII-RETIC (groups RTICC-RD12/0036/0001 and RTICC-RD12/0036/0021, respectively) and by CIBERONC (groups CB16/12/00352 and CB16/12/00273, respectively). Research co-financed by FEDER funds.S

The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras

Retraction in The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras. [J Biol Chem. 2018]Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.This work was supported in part by Programa General del Conocimiento (BMC2001-0057), Intramural Instituto de Salud Carlos III (ISCIII) (01/16), and SAF2003-02604 (Ministerio de Ciencia y Tecnología) grants (to J. M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

Shoc2/Sur8 Protein Regulates Neurite Outgrowth

The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.This work was supported by FIS grant (PI10/00815) to JLO; CIBERNED to MC; SAF2008-01951, Comunidad Autónoma de Madrid (CAM) S-SAL-0202-2006-01 and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) to TI; FIS grant PI12/00775 and ISCIII-RETIC (Red Temática de Investigación Cooperativa en Cáncer) RD12/0036/0027 from the Instituto de Salud Carlos III to PSG; and FIS grants (PI09/0562 and PI13/00703), ISCIII-RETIC (RD06/0020/0003 and RD12/0036/0021), and the Spanish Association Against Cancer (AECC) to JMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.JMR-C received grant support from MINECO-FEDER (SAF2016-78852-R), AESI-ISCIII (PI20CIII/00029) and Spanish Association against Cancer (AECC, CGB14143035THOM). ES group was supported by grants from ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-UIC-076), Areces Foundation (CIVP19A5942), Solorzano-Barruso Foundation (FS/32–2020) and by ISCIII-CIBERONC (group CB16/12/00352). Funding to AM group was provided by the Agencia Estatal de Investigación (PID2019-104867RB-I00/AEI/10.13039/501100011033) and by ISCIII-CIBERONC (group CB16/12/00273). TI was supported by grant PID2020-115218RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe” and by ISCIII-CIBERNED. RB received grant support from AESI-ISCIII (PI20CIII/00019). Finally, DP-J and MY groups were supported by grants 1.012.022 (to DP-J), 1.010.929 and 1.400.002 (both to MY) from Fundación Universidad Alfonso X el Sabio (FUAX). All research co-financed by FEDER funds.S

SOS GEFs in health and disease.

SOS1 and SOS2 are the most universal and widely expressed family of guanine exchange factors (GEFs) capable or activating RAS or RAC1 proteins in metazoan cells. SOS proteins contain a sequence of modular domains that are responsible for different intramolecular and intermolecular interactions modulating mechanisms of self-inhibition, allosteric activation and intracellular homeostasis. Despite their homology, analyses of SOS1/2-KO mice demonstrate functional prevalence of SOS1 over SOS2 in cellular processes including proliferation, migration, inflammation or maintenance of intracellular redox homeostasis, although some functional redundancy cannot be excluded, particularly at the organismal level. Specific SOS1 gain-of-function mutations have been identified in inherited RASopathies and various sporadic human cancers. SOS1 depletion reduces tumorigenesis mediated by RAS or RAC1 in mouse models and is associated with increased intracellular oxidative stress and mitochondrial dysfunction. Since WT RAS is essential for development of RAS-mutant tumors, the SOS GEFs may be considered as relevant biomarkers or therapy targets in RAS-dependent cancers. Inhibitors blocking SOS expression, intrinsic GEF activity, or productive SOS protein-protein interactions with cellular regulators and/or RAS/RAC targets have been recently developed and shown preclinical and clinical effectiveness blocking aberrant RAS signaling in RAS-driven and RTK-driven tumors.ES and FCB were supported by grants from ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-UIC 076) and Areces Foundation (CIVP19A5942). JMRC and NZ received grant support from MINECO-FEDER (SAF2016-78852-R and Spanish Association against Cancer (AECC/CGB14142035THOM). ES and JMRC were also supported by ISCIII-CIBERONC (groups CB16/12/00352 and CB16/12/00273, respectively). Research co-financed by FEDER funds.S

PGA(1)-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes

The cyclopentenone prostaglandin A(1) (PGA(1)) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA(1) triggers apoptosis by a process that entails the specific activation of H-and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA(1) treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118-S, defective for binding PGA(1), did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA(1) evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.We thank T O'Boyle and D Perez-Sala for the critical reading of this manuscript. APR, TG, and LMD were recipients of fellowships from the Comunidad de Madrid (APR), Ministerio de Educacion y Ciencia (TG), and FIS-BEFI (LMD). Grant support was awarded to JMRC from Fondo de Investigaciones Sanitarias-Intrasalud (PI09/0562 and PI13/00703) and the Spanish Association Against Cancer. JLO received grants from the Fondo de Investigaciones Sanitarias (CP07/00141 and PI10/00815). AM received grants from Ministerio de Economia y Competitividad of Spain-Fondo Europeo de Desarrollo Regional (FEDER) to AM (SAF2013-43468-R), Comunidad de Madrid to AM (S2010/BMD-2344 Colomics2) and Instituto de Salud Carlos III-FEDER. JMS received grants from the Fondo de Investigaciones Sanitarias (PI070356) and `Ayuda Intramural de Incorporacion al CSIC'. ES, AM, JMRC, and PC, respectively, received Grants RD06/0020/0000 and RD12/0036/0001, RD06/0020/0003 and RD12/0036/0021, and RD06/0020/0105 and RD12/0036/0033 from Instituto de San Carlos III-RETIC (Red Tematica de Investigacion Cooperativa en Cancer).S