ABSTRACT
ADP-ribosylation factors (Arfs) are Ras-like GTP-binding proteins that regulate membrane traffic and actin remodeling. Arf function requires GTP hydrolysis but Arf lacks GTPase activity; consequently, Arf function is dependent on Arf GTPase-activating proteins (GAPs). The Arf GAPs are a structurally diverse group of at least 16 proteins. Several Arf GAPs use a single Arf isoform. However, due to structural differences, the conditions supporting productive interactions between Arf and different Arf GAPs vary. Here, we describe preparation and basic properties of three Arf GAPs. We use these proteins to illustrate assays for Arf GAP activity. Conditions that optimize activity for each GAP are discussed. These methods can be used for the further characterization of Arf-Arf GAP interaction that is necessary for understanding the function of Arf in cellular physiology.
Subject(s)
ADP-Ribosylation Factors/analysis , Adaptor Proteins, Signal Transducing/analysis , GTP Phosphohydrolase Activators/analysis , GTPase-Activating Proteins/analysis , ADP-Ribosylation Factor 1/metabolism , Animals , Fluorescence , Guanosine Triphosphate/metabolism , Humans , Phosphorus Radioisotopes , Tryptophan/chemistryABSTRACT
BACKGROUND: Areca (named as betel) is an important etiological factor linked with the high prevalence of oral squamous cell carcinoma (OSCC) in South-Asian countries. This in vitro study investigated the cellular changes and signaling activation in oral keratinocytes in response to areca nut extract (ANE) treatment. METHODS: Normal human oral keratinocyte (NHOK) and oral epidermoid carcinoma cell, Meng-1 (OECM-1) OSCC cell line were treated with variable dosages of ripen ANE. The morphological and cytoskeletal changes, as well as the activation of GTPase proteins and signaling kinases, were analyzed. RESULTS: Most NHOK cells in culture were polygonal, with only <5% cells exhibiting fibroblastoid morphology. However, 10 microg/ml ANE elicited fibroblastoid morphological change, genesis of lamellipodia, loss of subcortical actin, and stress-fiber formation in approximately 25% cultivated NHOK cells. Similar morphological changes were observed in nearly all OECM-1 cells following the ANE treatment. The activation of Rac and Rho GTPase, together with the prominent phosphorylation of a stress-activated kinases, particularly JNK1, was identified in treated OECM-1 cells. CONCLUSION: The novel evidences from the study that ANE impairs the actin organization and activates the signals in oral keratinocytes might bestow further insight into the impacts of ANE in oral pathogenesis.
