Title

14-3-3 proteins in calcium-sensing receptor cell signalling

Abstract

The calcium-sensing receptor (CaR) maintains calcium homeostasis, but also influences processes such as cell proliferation, differentiation and apoptosis through activation of signalling pathways such as Rho and ERK1/2. To provide insight into mechanisms controlling CaR signalling, a yeast two-hybrid (Y2H) screen was performed using the CaR intracellular tail as bait. Several interacting proteins were identified including the 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, preferring targets containing phosphorylated motifs. They influence a multitude of cellular processes, including ERK1/2 signalling.

Co-immunoprecipitation (co-IP) and pulldown assays have confirmed CaR-14-3-3 in vivo and in vitro interaction, respectively, for 14-3-3 theta. Confocal microscopy has demonstrated that both 14-3-3 isoforms co-localise with the CaR in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923. A 14-3-3 consensus binding motif, Rx1-2Sx2-3S, exists within this region of the CaR tail where serine 895 is putatively phosphorylated. Site-directed mutagenesis and co-IP assays have shown that the serine 895 is not primarily responsible for mediating CaR-14-3-3 theta interaction. Further studies aim to establish the significance of the entire consensus motif for CaR-14-3-3 theta interaction.

To determine the role of 14-3-3 proteins in CaR-mediated ERK1/2 activity, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta, stimulated with extracellular calcium and analysed for ERK1/2 activity by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR. In addition, the possible role of 14-3-3 theta and zeta in CaR-mediated modulation of Rho-dependent stimulation of serum-response element transcription was examined in HEK293 cells using a luciferase assay. Preliminary results suggest that both 14-3-3 theta and zeta inhibit Rho signalling to a similar extent.

Differential binding of the two 14-3-3 isoforms to the CaR tail initially suggested differences in the way these isoforms may influence CaR-mediated signalling but experiments to date have not exposed such differences. The functional significance of the CaR-14-3-3 interaction will be further examined with other CaR-mediated signalling pathways.

Keywords

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