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

Shoc2/Sur8 protein regulates neurite outgrowth

This is an openaccess article distributed under the terms of the Creative Commons Attribution License.-- et al.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.GL, TG and LMD were recipients of fellowships from the Ministerio de Educación y Ciencia (MEC) (to GL, TG), and Fondo de Investigaciones Sanitarias (FIS) (to LMD). LSR held a postdoctoral research contract from CIBERNED. This work was supported by FIS grant (PI10/00815) to JLO; CIBERNED to MC; SAF2008-01951, Comunidad Autónoma de Madrid (CAM) SSAL-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), ISCIIIRETIC (RD06/0020/0003 and RD12/0036/0021), and the Spanish Association Against Cancer (AECC) to JMR.Peer Reviewe

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

Caracterización del gen de la enfermedad de Lafora: construcción de un mapa físico de alta resolución y definición de los candidatos posicionales

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-01-200

Caracterizacion del gen de la enfermedad de Lafora Construccion de un mapa fisico de alta resolucion y definicion de los candidatos posicionales

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

Empatía autopercibida en tutores y residentes de una unidad docente de atención familiar y comunitaria.

Introduction: Empathy is an essential element of medical professionalism, recognized as one of the main components of health care because of its importance in the doctor-patient relationship, its presence is important in health professionals, especially in tutors of different training programs. Objectives: To assess the degree of empathy towards patients of tutors and residents of family medicine and family nursing teaching unit in Tenerife (Canary Islands, Spain) and its association with sociodemographic factors. Material and Method: Descriptive, cross-sectional and analytical study. The population was composed of tutors and residents of Family and Community Medicine and Nursing of the Multiprofessional Teaching Unit of Family and Community Care Tenerife Zone I. Taking advantage of a training meeting, they were invited to complete a self-administered questionnaire that contained the following variables: self-perceived empathy measured using the Jefferson scale, validated in Spanish that has shown high validity and reliability (score between 20 and 140). In addition, gender, age, country of origin, type of professional (tutor or resident), specialty, years of work experience, training year and area of work were collected. The sample was finally composed of 76 tutors and 60 residents. Results: A total of 136 participants participated, 77.6% were women and 91.7% were born in Spain. The mean age was 39.21 years (SD: 11.76). The global empathy mean was 121.97 (SD: 11.06). No differences were found by specialization: Family Medicine tutors 122.94 (SD: 10.73) versus Family Nursing tutors 120.65 (SD: 13.81); p= 0.450. Family Medicine residents 120.96 (SD: 10.69) versus Family Nursing residents 127.40 (SD: 4.87); p= 0.178. Likewise, no association was found between empathy and the condition of being a tutor or resident or with the rest of the variables studied. Conclusions: We consider that the level of empathy with patients in tutors and residents of the Teaching Unit of Family and Community Care of Tenerife zone I is high, finding no statistically significant differences with any of the analyzed variables.Introducción: La empatía es un elemento esencial del profesionalismo médico, reconocido como uno de los componentes principales de la atención sanitaria por su importancia en la relación médico-paciente, siendo importante que esté presente en profesionales sanitarios, especialmente en los tutores de los distintos programas formativos. Objetivos: Valorar el grado de empatía con los pacientes de tutores y residentes de Medicina Familiar y Comunitaria y Enfermería Familiar y Comunitaria de una Unidad Docente Multiprofesional de Tenerife (Islas Canarias, España), y su asociación con factores sociodemográficos. Material y Método: Estudio descriptivo transversal y analítico. La población estuvo compuesta por tutores y residentes de Medicina y Enfermería Familiar y Comunitaria de la Unidad Docente Multiprofesional de Atención Familiar y Comunitaria Tenerife Zona I. Aprovechando un encuentro formativo, se les invitó a completar un cuestionario autoadministrado que contenía las siguientes variables: empatía autopercibida medida mediante la escala de Jefferson, validada al español que ha mostrado alta validez y confiabilidad, pudiendo obtenerse una puntuación entre 20 y 140. Además, se recogió género, edad, país de origen, tipo de profesional (tutor o residente), especialidad, años de experiencia laboral, año formativo y ámbito de trabajo. La muestra finalmente estuvo compuesta por 76 tutores y 60 residentes. Resultados: Participaron un total de 136 profesionales. Un 77,6% eran mujeres y 91,7% habían nacido en España. La edad media fue 39,21 años (DT: 11,76). La media de empatía global fue 121,97 (DT: 11,06). No se encontraron diferencias por especialidad: tutores de Medicina Familiar 122,94 (DT: 10,73) versus tutores de Enfermería Familiar 120,65 (DT: 13,81); p = 0,450. Residentes de Medicina Familiar 120,96 (DT: 10,69) versus residentes de Enfermería Familiar 127,40 (DT: 4,87); p = 0,178. Asimismo, tampoco se encontró asociación entre la empatía y la condición de ser tutor o residente ni con el resto de las variables estudiadas. Conclusiones: Consideramos que el nivel de empatía con los pacientes en los tutores y residentes de Medicina y Enfermería Familiar y Comunitaria de la Unidad Docente Multiprofesional de Atención Familiar y Comunitaria de Tenerife Zona I es alto, no encontrando diferencias estadísticamente significativas con ninguna de las variables analizadas

A Novel Protein Tyrosine Phosphatase Gene Is Mutated in Progressive Myoclonus Epilepsy of the Lafora Type (EPM2)

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23-q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microeletion within the EPM2 critical region, present in homozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2, presents alternative splicing in the 5′ and 3′ end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolis

Shoc2 depletion reduces phospho-ERK and phospho-MEK levels induced by RTK activation.

<p>HEK293T cells were transiently transfected (100 nM) with either control siRNA (siRNA-Control) or human Shoc2-specific siRNA (siRNA-Shoc2). At 48 h post-transfection, cells were serum-starved for 18 h and then incubated with vehicle (0), or 5-25 ng/ml FGF for 10 min. (<b>A</b>), endogenous Shoc2 expression was assessed, from the cell lysates, by immunoblot using anti-Shoc2 rabbit polyclonal antibody and normalized to tubulin levels, expressed as a percentage (corresponding to the mean of five separate assays, in all cases with a SD ≤10% of the mean). The specific effect of the siRNA-Shoc2 was tested throughout detection of Grb2, Ras, C-Raf, ERK and MEK protein levels, by immunoblot of the cell lysates with the corresponding antibodies. Cell lysates were also analyzed by immunoblot using anti-p-ERK/ERK, and anti-p-MEK/MEK antibodies (bottom panels); in each case, fold increase denotes the ratio p-ERK/ERK, and p-MEK/MEK levels estimated as the mean of five separate assays (in all cases with a SD ≤10% of the mean). (<b>B</b>), the expression of Shoc2 was assessed by qRT-PCR. Data represents the mean ± SD of four independent experiments. The detection of GAPDH (normalization control) was performed using specific primers. Results were calculated by the 2-^ΔΔCT method. *P≤0.001 compared with siRNA-control; bars show SD.</p