Project description:Endocannabinoids are among the most powerful modulators of synaptic transmission throughout the nervous system, and yet little is understood about the release of endocannabinoids from postsynaptic compartments. Here we report an unexpected finding that endocannabinoid release requires synucleins, key contributors to Parkinson's disease. We show that endocannabinoids are released postsynaptically by a synuclein-dependent and SNARE-dependent mechanism. Specifically, we found that synuclein deletion blocks endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wild-type but not of mutant α-synuclein. Whole-cell recordings and direct optical monitoring of endocannabinoid signaling suggest that the synuclein deletion specifically blocks endocannabinoid release. Given the presynaptic role of synucleins in regulating vesicle lifecycle, we hypothesize that endocannabinoids are released via a membrane interaction mechanism. Consistent with this hypothesis, postsynaptic expression of tetanus toxin light chain, which cleaves synaptobrevin SNAREs, also blocks endocannabinoid-dependent signaling. The unexpected finding that endocannabinoids are released via a synuclein-dependent mechanism is consistent with a general function of synucleins in membrane trafficking and adds a piece to the longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.
Project description:Deregulated RTK activity has been implicated as a causal leukemogenic factor in the context of molecular aberrations that perturb differentiation in the hematopoietic lineage such as in childhood ALL. A deeper understanding of RTK signaling processes on a system-wide scale will be key in defining critical components of signaling networks. To link RTK activity with in vivo output in primary ALL we took a functional approach, which combined SH2 domain binding, mass spectrometry, and transcriptome analyses. Structure and composition of evolving networks were highly diverse with few generic features determined by receptor and cell type. A combinatorial assembly of varying context-dependent and few generic signaling components at multiple levels likely generates output specificity. PAK2 was identified as a phosphoregulated FLT3 target, whose allosteric inhibition resulted in apoptosis of ALL cells. Our studies provide evidence that a functional approach to leukemia signaling may yield valuable information for a network-directed intervention. Three different primary ALL samples expressing FLT3 and/or PDGFRß were stimulated with the corresponding ligands FLT3L and PDGF-BB for five time points. Unstimulated samples served as control. ALL22089 coexpresses FLT3 and PDGFRß. ALL109 expresses PDGFRß. ALL114 expresses FLT3.
Project description:Deregulated RTK activity has been implicated as a causal leukemogenic factor in the context of molecular aberrations that perturb differentiation in the hematopoietic lineage such as in childhood ALL. A deeper understanding of RTK signaling processes on a system-wide scale will be key in defining critical components of signaling networks. To link RTK activity with in vivo output in primary ALL we took a functional approach, which combined SH2 domain binding, mass spectrometry, and transcriptome analyses. Structure and composition of evolving networks were highly diverse with few generic features determined by receptor and cell type. A combinatorial assembly of varying context-dependent and few generic signaling components at multiple levels likely generates output specificity. PAK2 was identified as a phosphoregulated FLT3 target, whose allosteric inhibition resulted in apoptosis of ALL cells. Our studies provide evidence that a functional approach to leukemia signaling may yield valuable information for a network-directed intervention. Primary ALL samples were investigated on Affymetrix SNP 6.0 arrays for copy-number changes related to receptor tyrosine kinases (RTKs). Buffy-Coat samples of healthy persons were used as reference.
Project description:While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III (Endosomal Sorting Complex Required for Transport-III), were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding loop, and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound to N-Ras but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked epidermal growth factor receptor (EGFR) recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.
Project description:NKX3.1 is a homeobox gene that codes for a haploinsufficient prostate cancer tumor suppressor. NKX3.1 protein levels are down-regulated in the majority of primary prostate cancer tissues. NKX3.1 expression in PC-3 cells increased insulin-like growth factor binding protein-3 (IGFBP-3) mRNA expression 10-fold as determined by expression microarray analysis. In both stably and transiently transfected PC-3 cells and in LNCaP cells, NKX3.1 expression increased IGFBP-3 mRNA and protein expression. In prostates of Nkx3.1 gene-targeted mice Igfbp-3 mRNA levels correlated with Nkx3.1 copy number. NKX3.1 expression in PC-3 cells attenuated the ability of insulin-like growth factor-I (IGF-I) to induce phosphorylation of type I IGF receptor (IGF-IR), insulin receptor substrate 1, phosphatidylinositol 3-kinase, and AKT. The effect of NKX3.1 on IGF-I signaling was not seen when cells were exposed to long-R3-IGF-I, an IGF-I variant peptide that does not bind to IGFBP-3. Additionally, small interfering RNA-induced knockdown of IGFBP-3 expression partially reversed the attenuation of IGF-IR signaling by NKX3.1 and abrogated NKX3.1 suppression of PC-3 cell proliferation. Thus, there is a close relationship in vitro and in vivo between NKX3.1 and IGFBP-3. The growth-suppressive effects of NKX3.1 in prostate cells are mediated, in part, by activation of IGFBP-3 expression.
Project description:Ion channels modulate ion flux across cell membranes, activate signal transduction pathways, and influence cellular transport-vital biological functions that are inexorably linked to cellular processes that go awry during carcinogenesis. Indeed, deregulation of ion channel function has been implicated in cancer-related phenomena such as unrestrained cell proliferation and apoptotic evasion. As the prototype for ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs) have been extensively studied in the context of neuronal cells but accumulating evidence also indicate a role for nAChRs in carcinogenesis. Recently, variants in the nAChR genes CHRNA3, CHRNA5, and CHRNB4 have been implicated in nicotine dependence and lung cancer susceptibility. Here, we silenced the expression of these three genes to investigate their function in lung cancer. We show that these genes are necessary for the viability of small cell lung carcinomas (SCLC), the most aggressive type of lung cancer. Furthermore, we show that nicotine promotes SCLC cell viability whereas an α3β4-selective antagonist, α-conotoxin AuIB, inhibits it. Our findings posit a mechanism whereby signaling via α3/α5/β4-containing nAChRs promotes lung carcinogenesis.
Project description:Deregulated RTK activity has been implicated as a causal leukemogenic factor in the context of molecular aberrations that perturb differentiation in the hematopoietic lineage such as in childhood ALL. A deeper understanding of RTK signaling processes on a system-wide scale will be key in defining critical components of signaling networks. To link RTK activity with in vivo output in primary ALL we took a functional approach, which combined SH2 domain binding, mass spectrometry, and transcriptome analyses. Structure and composition of evolving networks were highly diverse with few generic features determined by receptor and cell type. A combinatorial assembly of varying context-dependent and few generic signaling components at multiple levels likely generates output specificity. PAK2 was identified as a phosphoregulated FLT3 target, whose allosteric inhibition resulted in apoptosis of ALL cells. Our studies provide evidence that a functional approach to leukemia signaling may yield valuable information for a network-directed intervention.
Project description:Deregulated RTK activity has been implicated as a causal leukemogenic factor in the context of molecular aberrations that perturb differentiation in the hematopoietic lineage such as in childhood ALL. A deeper understanding of RTK signaling processes on a system-wide scale will be key in defining critical components of signaling networks. To link RTK activity with in vivo output in primary ALL we took a functional approach, which combined SH2 domain binding, mass spectrometry, and transcriptome analyses. Structure and composition of evolving networks were highly diverse with few generic features determined by receptor and cell type. A combinatorial assembly of varying context-dependent and few generic signaling components at multiple levels likely generates output specificity. PAK2 was identified as a phosphoregulated FLT3 target, whose allosteric inhibition resulted in apoptosis of ALL cells. Our studies provide evidence that a functional approach to leukemia signaling may yield valuable information for a network-directed intervention.