Effect of Sumoylation on Gsk-3beta-Mediated Autophagy

Project: A - Government Institutionb - Ministry of Science and Technology

Description

Autophagy, a type II cell death, is an evolutionarily conserved mechanism for the degradation of cellular components in the cytoplasm. It is considered that the process of autophagy is composing of many signal pathways. However, the detail mechanism in mammalian cells is unclear. Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that phosphate and inactivate glycogen kinase, which is involved in the apoptosis induced by cytotoxic agents in various cells. Nevertheless, the scenario of GSK-3β in conducting a promising process of autophagy is still obscure. Small ubiquitin-like modifier (SUMO) modification is considered as an important control in protein activation, translocation, and degradation. Recently, SUMOylation is demonstrated to be involved in GSK-3β regulation. However, the detail mechanism and the role of SUMOylation in regulation of autophagy are unclear. In the present study, using cadmium (Cd)-treated mesangial cells as experimental models, we plan to investigate the the molecular mechanism underlying GSK-3β-mediated autophagy, and to explore the role of SUMOylation in the autophagy. Our preliminary results has proven that the proportion of autophagy was increased after treatment with cadmium for 24 h. In addition, a parallel experiment indicated inhibition of GSK-3β by inhibitor or siRNA knockdown decreased Cd-induced autophagy, suggesting GSK-3β may play a role in the induction of Cd-induced autophagy. Following this line, we plan to investigate the molecular mechanism of GSK-3β−mediated autophagy in mesangial cells. YEAR-1: Specific Aim: To examine the GSK-3β and SUMOylation in regulating autophagy. Hypothesis: Cd-induced autophagy is through SUMOylation-GSK-3β signaling pathway. 1.1. To test whether Cd are able to induce autophagy in MES-13 mesangial cells using flow cytometry, EM and immunoblotting to detect acidic AVs, membranous AVs, and LC3, respectively. 1.2. To test whether GSK-3β is involved in Cd-induced autophagy using siRNA to knock down the expression of GSK-3β or transfection the CMV-driven GSK-3β to over-express GSK-3β protein, and then detect the proportion of autophagy. 1.3. Site-directed mutagenesis on Ser-9 and/or Tyr-216 of GSK-3β to test their effect on autophagy. 1.4. To assess the role of SUMOylation in autophagy using immunoblotting to detect the expression of SUMO-1, 2, 3 and 4 or using siRNA to knockdown the expression of SUMO-1, 2, 3 and 4 to detect the percentage of autophagy. 1.5. To assess the interaction between SUMOylation and GSK-3β using siRNA to knockdown the expression of SUMO-1, 2, 3 and 4 to detect the activity of GSK-3β and the formation of complex of GSK-3β and SUMO proteins. YEAR-2: Specific Aim: To investigate the molecular mechanism of GSK-3β in Cd-induced autophagy. Hypothesis: NF-κB and CREB are involved in GSK-3β-mediatd autophagy. 2.1. To assess the role of NF-κB and CREB in Cd-induced autophagy using EMSA assay to detect the activity of NF-κB and CREB. 2.2 To assess the role of GSK-3β in activation of NF-κB and CREB in Cd-indcued autophagy using inhibitor or siRNA knockdown of GSK-3β to detect the activity of NF-κB and CREB. 2.3 To assess the role of SUMOylation in Cd-indcued NF-κB and CREB activation using siRNA knockdown of SUMO-1, 2, 3 and 4 to detect the activity of NF-κB and CREB. YEAR-3: Specific Aim: To determine the effects of GSK-3β in GSK-3β knockout embryonic fibroblasts. Hypothesis: Cd-induced autophagy is through GSK-3β in GSK-3β knockout embryonic fibroblasts 3.1. To establish GSK-3β knockout embryonic fibroblasts. 3.2. To assess the role of GSK-3β in Cd-induced autophagy. 3.3. To assess the role of SUMOylation in regulating autophagy using siRNA knockdown of SUMO-1, 2, 3 and 4 to detect the proportion of autophagy. 3.4. To assess the role of NF-κB and CREB in regulating autophagy using siRNA knockdown of NF-κB and CREB to detect the proportion of autophagy. Complete this study will expand our knowledge inCd-induced nephrotoxicity.
StatusFinished
Effective start/end date8/1/127/31/13