Dataset Information

OSBP-related protein 4L promotes phospholipase C?3 translocation from the nucleus to the plasma membrane in Jurkat T-cells.

ABSTRACT: Phosphoinositide phospholipases C (PLCs) are a family of eukaryotic intracellular enzymes with important roles in signal transduction. In addition to their location at the plasma membrane, PLCs also exist within the cell nucleus where they are stored. We previously demonstrated that OSBP-related protein 4L (ORP4L) anchors cluster of differentiation 3? (CD3?) to the heterotrimeric G protein subunit (G?_q/11) to control PLC?3 relocation and activation. However, the underlying mechanism by which ORP4L facilitates PLC?3 translocation remains unknown. Here, using confocal immunofluorescence microscopy and coimmunoprecipitation assays, we report that ORP4L stimulates PLC?3 translocation from the nucleus to the plasma membrane in Jurkat T-cells in two steps. First, we found that ORP4L is required for the activation of Ras-related nuclear protein (RAN), a GTP-binding nuclear protein that binds to exportin 1 and eventually promotes the nuclear export of PLC?3. Second, we also observed that ORP4L interacts with vesicle-associated membrane protein-associated protein A (VAPA) through its two phenylalanines in an acidic tract (FFAT) motif. This complex enabled PLC?3 movement to the plasma membrane, indicating that PLC?3 translocation occurs in a VAPA-dependent manner. This study reveals detailed mechanistic insight into the role of ORP4L in PLC?3 redistribution from storage within the nucleus to the plasma membrane via RAN activation and interaction with VAPA in Jurkat T-cells.

SUBMITTER: Pan G

PROVIDER: S-EPMC6231138 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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OSBP-related protein 4L promotes phospholipase Cβ3 translocation from the nucleus to the plasma membrane in Jurkat T-cells.

Pan Guoping G Cao Xiuye X Liu Bo B Li Chaowen C Li Dan D Zheng Jie J Lai Chaofeng C Olkkonen Vesa M VM Zhong Wenbin W Yan Daoguang D

The Journal of biological chemistry 20180920 45

Phosphoinositide phospholipases C (PLCs) are a family of eukaryotic intracellular enzymes with important roles in signal transduction. In addition to their location at the plasma membrane, PLCs also exist within the cell nucleus where they are stored. We previously demonstrated that OSBP-related protein 4L (ORP4L) anchors cluster of differentiation 3ϵ (CD3ϵ) to the heterotrimeric G protein subunit (Gα<sub>q/11</sub>) to control PLCβ3 relocation and activation. However, the underlying mechanism b ...[more]

PMID: 30237164

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Project description:BackgroundCopines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA - cpnF.ResultsIndependent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P.ConclusionsOur studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.

Dataset Information

OSBP-related protein 4L promotes phospholipase C?3 translocation from the nucleus to the plasma membrane in Jurkat T-cells.

Publications

OSBP-related protein 4L promotes phospholipase Cβ3 translocation from the nucleus to the plasma membrane in Jurkat T-cells.

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