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Pozuelo-Rubio M.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa
Cell Death and Differentiation | Year: 2011

14-3-3s are binding proteins with survival functions in cells by interaction with proteins involved in the regulation of cell fate. The role of 14-3-3 during autophagy was investigated, thus, a forced expression of 14-3-3 reduces C2-ceramide-induced autophagy, whereas depletion of 14-3-3 promotes autophagy. The 14-3-3 role in autophagyc-related proteins was also investigated. The human vacuolar protein sorting 34 (hVps34), the class III phosphatidylinositol-3-kinase mediates multiple vesicle-trafficking processes such as endocytosis and autophagy, its activation being a requirement for autophagy initiation. Using chromatography techniques, hVps34 were eluted from a 14-3-3 affinity column, showing also a direct interaction with 14-3-3 proteins under physiological condition. Further analysis suggests that hVps34/14-3-3 association is a phorbol-12-myristate-13-acetate-dependent phosphorylated mechanism promoting a strong inhibition of the hVps34 lipid kinase activity, proteins kinase C being the likely kinase involved in phosphorylation and 14-3-3 binding of hVps34 under physiological conditions. Meanwhile, stimulation of autophagy leads to the dissociation of the 14-3-3/hVps34 complex enhancing hVps34 lipid kinase activity. Forced expression of 14-3-3 reduces hVps34 kinase activity and depletion of 14-3-3 promotes upregulation of this activity. In this study, 14-3-3 proteins are shown as a negative regulator of autophagy through regulation of a key component of early stages of the autophagy pathway, such as hVps34. © 2011 Macmillan Publishers Limited All rights reserved.


Simon-Areces J.,Instituto Cajal | Dopazo A.,Genomics Unit | Dettenhofer M.,Harvard University | Rodriguez-Tebar A.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa | And 2 more authors.
PLoS ONE | Year: 2011

Neurogenin3, a proneural transcription factor controlled by Notch receptor, has been recently shown to regulate dendritogenesis and synaptogenesis in mouse hippocampal neurons. However, little is known about the molecular mechanisms involved in these actions of Ngn3. We have used a microarray analysis to identify Ngn3 regulated genes related with cytoskeleton dynamics. One of such genes is Fmn1, whose protein, Formin1, is associated with actin and microtubule cytoskeleton. Overexpression of the Fmn1 isoform-Ib in cultured mouse hippocampal neurons induced an increase in the number of primary dendrites and in the number of glutamatergic synaptic inputs at 4 days in vitro. The same changes were provoked by overexpression of Ngn3. In addition downregulation of Fmn1 by the use of Fmn1-siRNAs impaired such morphological and synaptic changes induced by Ngn3 overexpression in neurons. These results reveal a previously unknown involvement of Formin1 in dendritogenesis and synaptogenesis and indicate that this protein is a key component of the Ngn3 signaling pathway that controls neuronal differentiation. © 2011 Simon-Areces et al.


Pozuelo-Rubio M.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa
FEBS Journal | Year: 2010

14-3-3 is a family of proteins comprising several isoforms that, in many cases, promote cell survival by association with proapoptotic proteins. This study was designed to obtain further understanding of the 14-3-3 role in apoptosis regulation, by analyzing apoptosis-related protein-14-3-3 interactions. Western blot analysis of an eluted fraction from the 14-3-3-affinity chromatography column identified proapoptotic proteins as receptor-interacting protein 3 and Bcl-2-antagonist/killer as new phophorylation-dependent 14-3-3-binding proteins under physiological conditions. The apoptosis inducer C2-ceramide promoted decay of the 14-3-3-binding signal of protein cell extracts. Investigation of the role of 14-3-3 in C2-ceramide-induced apoptosis showed that depletion of the 14-3-3ζ isoform sensitized to cell death, whereas overexpression of this isoform delayed cell death. A combination of tandem affinity purification and liquid chromatography-tandem MS techniques identified 15 proteins involved in cell survival processes whose 14-3-3-binding status changed during C2-ceramide-induced apoptosis. Under physiological conditions, desmin was clearly identified as a new 14-3-3-interactor protein, and vasodilator- stimulated phosphoprotein, nucleophosmin and calmodulin, whose 14-3-3 binding was suggested by others on the basis of MS analysis, were confirmed here as phosphorylation-dependent 14-3-3-associated proteins. Interestingly, proteins related to the regulation of DNA double-strand break repair in the early stages of apoptosis, such as DNA-dependent protein kinase, or the regulation of cell shrinkage during apoptosis, such as vasodilator-stimulated phosphoprotein and death promoters like receptor-interacting protein 3, were identified as 14-3-3-associated proteins whose 14-3-3-binding status changed when apoptosis was initiated. The functional diversity of these identified proteins suggests that 14-3-3 may regulate the apoptotic process through new mechanisms, in addition to others previously characterized. Structured digital abstract A list of the large number of protein-protein interactions described in this article is available via the MINT article ID © 2010 The Author Journal compilation.


Chacon P.J.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa | Arevalo M.A.,Instituto Cajal Of Neurobiologia | Tebar A.R.,Centro Andaluz Of Biologia Molecular Y Medicina Regenerativa
Molecular and Cellular Neuroscience | Year: 2010

NGF diminishes dendrite complexity in cultured hippocampal neurons by decreasing the number of primary and secondary dendrites, while increasing the length of those that remain. The transduction pathway used by NGF to provoke dendrite elongation involves the activation of NF-κ-B and the expression of the homologues of Enhancer-of-split 1 gene. Here, we define important steps that link NGF with NF-κ-B activation, through the activity of protein tyrosine phosphatase 1B (PTP1B). Binding of NGF to p75NTR stimulates PTP1B activity, which can be blocked by either pharmacological inhibition of the phosphatase or by transfecting neurons with a dn PTP1B isoform, whereby NGF is no longer able to stimulate dendrite growth. Indeed, overexpressing PTP1B alone provoked dendrite growth and further studies revealed a role for the src kinase downstream of PTP1B. Again, loss of src activity largely cancelled out the capacity of NGF to promote dendrite growth, whereas overexpression of v-src in neurons was sufficient to promote dendrite growth. Finally, the NGF/p75NTR/PTP1B/src kinase pathway led to the tyrosine phosphorylation of I-κ-Bα prior to its degradation, an event that is necessary for NF-κ-B activation. Indeed, the dendrite growth response to NGF was lost when neurons were transfected with a mutant form of I-κ-Bα that lacks tyr42. Thus, our data suggest that PTP1B fulfils a central role in the NGF signalling that controls dendrite patterning in hippocampal neurons. © 2010 Elsevier Inc. All rights reserved.


Alvarez-Perez J.C.,Mount Sinai School of Medicine | Ernst S.,University of Pittsburgh | Demirci C.,University of Pittsburgh | Demirci C.,Connecticut Childrens Medical Center | And 6 more authors.
Diabetes | Year: 2014

Hepatocyte growth factor (HGF) is a mitogen required for β-cell replication during pregnancy. To determine whether HGF/c-Met signaling is required for β-cell regeneration, we characterized mice with pancreatic deletion of the HGF receptor, c-Met (PancMet KO mice), in two models of reduced β-cell mass and regeneration: multiple low-dose streptozotocin (MLDS) and partial pancreatectomy (Ppx). We also analyzed whether HGF administration could accelerate β-cell regeneration in wild-type (WT) mice after Ppx. Mouse islets obtained 7 days post-Ppx displayed significantly increased c-Met, suggesting a potential role for HGF/c-Met in β-cell proliferation in situations of reduced β-cell mass. Indeed, adult PancMet KO mice displayed markedly reduced β-cell replication compared with WT mice 7 days post-Ppx. Similarly, β-cell proliferation was decreased in PancMet KO mice in the MLDS mouse model. The decrease in β-cell proliferation post-Ppx correlated with a striking decrease in D-cyclin levels. Importantly, PancMet KO mice showed significantly diminished β-cell mass, decreased glucose tolerance, and impaired insulin secretion compared with WT mice 28 days post-Ppx. Conversely, HGF administration in WT Ppx mice further accelerated β-cell regeneration. These results indicate that HGF/c-Met signaling is critical for β-cell proliferation in situations of diminished β-cell mass and suggest that activation of this pathway can enhance β-cell regeneration. © 2014 by the American Diabetes Association.

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