RESUMO
Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Proteínas 14-3-3/genética , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência Consenso , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hipocótilo/genética , Luz , Fosforilação , Fototropismo/efeitos da radiação , Ligação Proteica/efeitos da radiação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST, the epithelial cell adhesion molecule (EPCAM), the transcription factors SNAI1 (SNAIL) and SNAI2 (SLUG) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/patologia , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Fator de Necrose Tumoral alfa/farmacologia , Antineoplásicos Imunológicos/farmacologia , Apoptose , Proliferação de Células , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , NF-kappa B/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Células Tumorais CultivadasRESUMO
A signalling pathway involving PLEKHG5 (guanine exchange factor) for the Ras superfamily member RAB26 to transcription factor NF-κB was discovered in autophagy. PLEKHG5 was reported in glioblastoma multiforme (GBM) and correlates with patient survival. Thus, the generation of a cellular model for understanding PLEKHG5 signalling is the study purpose. We generated a CRISPR/Cas9-mediated knockout of PLEKHG5 in U251-MG glioblastoma cells and analysed resulting changes. Next, we used a mRFP-GFP-LC3+ reporter for visualisation of autophagic defects and rescued the phenotype of PLEKHG5 wildtype via transduction of a constitutively active RAB26QL-plasmid. Effects of overexpressing RAB26 were investigated and correlated with the O6-methylguanine-DNA methyltransferase (MGMT) and cellular survival. PLEKHG5 knockout showed changes in morphology, loss of filopodia and higher population doubling times. Accumulation of autolysosomes was resulted by decreased LAMP-1 in PLEKHG5-deficient cells. Rescue of PLEKHG5-/- restored the downregulation of RhoA activity, showed faster response to tumour necrosis factor and better cellular fitness. MGMT expression was activated after RAB26 overexpression compared to non-transduced cells. Survival of PLEKHG5 knockout was rescued together with sensitivity to temozolomide by RAB26QL. This study provides new insights in the PLEKHG5/RAB26 signalling within U251-MG cells, which suggests potential therapeutic strategies in other glioma cells and further in primary GBM.
