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1.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544135

RESUMO

Using model organisms to identify novel therapeutic targets is frequently constrained by pre-existing genetic toolkits. To expedite positive selection for identification of novel downstream effectors, we engineered conditional expression of activated CED-10/Rac to disrupt Caenorhabditis elegans embryonic morphogenesis, titrated to 100% lethality. The strategy of engineering thresholds for positive selection using experimental animals was validated with pharmacological and genetic suppression and is generalizable to diverse molecular processes and experimental systems.


Assuntos
Proteínas de Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética
2.
Mol Cancer Res ; 14(10): 1009-1018, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27418645

RESUMO

Recently, we identified that PREX1 overexpression is critical for metastatic but not tumorigenic growth in a mouse model of NRAS-driven melanoma. In addition, a PREX1 gene signature correlated with and was dependent on ERK MAPK activation in human melanoma cell lines. In the current study, the underlying mechanism of PREX1 overexpression in human melanoma was assessed. PREX1 protein levels were increased in melanoma tumor tissues and cell lines compared with benign nevi and normal melanocytes, respectively. Suppression of PREX1 by siRNA impaired invasion but not proliferation in vitro PREX1-dependent invasion was attributable to PREX1-mediated activation of the small GTPase RAC1 but not the related small GTPase CDC42. Pharmacologic inhibition of ERK signaling reduced PREX1 gene transcription and additionally regulated PREX1 protein stability. This ERK-dependent upregulation of PREX1 in melanoma, due to both increased gene transcription and protein stability, contrasts with the mechanisms identified in breast and prostate cancers, in which PREX1 overexpression was driven by gene amplification and HDAC-mediated gene transcription, respectively. Thus, although PREX1 expression is aberrantly upregulated and regulates RAC1 activity and invasion in these three different tumor types, the mechanisms of its upregulation are distinct and context dependent. IMPLICATIONS: This study identifies an ERK-dependent mechanism that drives PREX1 upregulation and subsequent RAC1-dependent invasion in BRAF- and NRAS-mutant melanoma. Mol Cancer Res; 14(10); 1009-18. ©2016 AACR.


Assuntos
GTP Fosfo-Hidrolases/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Melanoma/genética , Proteínas de Membrana/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Indazóis/farmacologia , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases , Masculino , Melanoma/metabolismo , Camundongos , Piperazinas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Sulfonamidas/farmacologia , Regulação para Cima , Vemurafenib
3.
J Biol Chem ; 283(29): 20600-11, 2008 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-18474593

RESUMO

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.


Assuntos
Fibrinolisina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Receptor PAR-1/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Ativação Enzimática , Humanos , Magnésio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor PAR-1/deficiência , Receptor PAR-1/genética , Transdução de Sinais/efeitos dos fármacos
4.
J Biol Chem ; 282(35): 25199-212, 2007 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-17620339

RESUMO

Plasma membrane targeting of G protein alpha (Galpha) subunits is essential for competent receptor-to-G protein signaling. Many Galpha are tethered to the plasma membrane by covalent lipid modifications at their N terminus. Additionally, it is hypothesized that Gq family members (Gqalpha,G11alpha,G14alpha, and G16alpha) in particular utilize a polybasic sequence of amino acids in their N terminus to promote membrane attachment and protein palmitoylation. However, this hypothesis has not been tested, and nothing is known about other mechanisms that control subcellular localization and signaling properties of G14alpha and G16alpha. Here we report critical biochemical factors that mediate membrane attachment and signaling function of G14alpha and G16alpha. We find that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences in their N termini and that the polycysteine sequence along with the adjacent polybasic region are both important for G16alpha-mediated signaling at the plasma membrane. Surprisingly, the isolated N termini of G14alpha and G16alpha expressed as peptides fused to enhanced green fluorescent protein each exhibit differential requirements for palmitoylation and membrane targeting; individual cysteine residues, but not the polybasic regions, determine lipid modification and subcellular localization. However, full-length G16alpha, more so than G14alpha, displays a functional dependence on single cysteines for membrane localization and activity, and its full signaling potential depends on the integrity of the polybasic sequence. Together, these findings indicate that G14alpha and G16alpha are palmitoylated at distinct polycysteine sequences, and that the adjacent polybasic domain is not required for Galpha palmitoylation but is important for localization and functional activity of heterotrimeric G proteins.


Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transdução de Sinais/fisiologia , Membrana Celular/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Peptídeos/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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