Title

The 14-3-3 isoforms theta and zeta bind the calcium-sensing receptor and modulate receptor signalling through the Rho pathway, but not the ERK1/2 pathway

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 cell signalling.

Co-immunoprecipitation (co-IP) studies have confirmed CaR-14-3-3 in vivo interaction for both 14-3-3 theta and zeta. Confocal microscopy experiments have demonstrated that both 14-3-3 isoforms co-localise with the CaR most likely in the endoplasmic reticulum. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail, which overlaps the filamin interaction domain. 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 phosphorylation by Western blot analysis. Neither 14-3-3 isoform modulated ERK1/2 activity through the CaR.

To determine the possible role of 14-3-3 theta or zeta in CaR-mediated Rho signalling, HEK-293 cells stably expressing the CaR were transfected with 14-3-3 theta or zeta and a luciferase reporter gene which allowed for the measurement of luciferase activity through Rho-dependent stimulation of serum-response element transcription. Results demonstrated that both 14-3-3 theta and zeta activated Rho signalling.

The enhancement of CaR-mediated Rho signalling by 14-3-3 isoforms suggests a general role for 14-3-3 proteins as chaperones in the regulation of Rho signalling, and possibly other CaR-mediated signalling pathways.

Keywords

Oral Presentation, Abstract only

Comments

Poster and oral presentation