Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1

Abstract

The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, we identified stromal interaction molecule 1 precursor (STIM1) as an auxiliary protein of this epithelial Ca2+channel using confocal microscopy analysis and GST pull-down assay. The STIM1 protein associates specifically with the C-terminal tail of TRPV4 to form a complex. In previous reports, we demonstrated that the serine824 residue of TRPV4 is one of the target phosphorylation sites of serum/glucocorticoid regulated kinase 1 (SGK1). In this report we further identified the role of serine 824 phosphorylation. The TRPV4 mutant S824D (not S824A) exhibited a diminished capacity to bind STIM1. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STIM1 is part of the TRPV4 protein complex. Our observations clearly suggest that the formation of a complex between TRPV4 and STIM1 and its plasma membrane localization are regulated through phosphorylation of serine824 of TRPV4, and that the STIM1-TRPV4 complex plays crucial roles in routing TRPV4 to the plasma membrane from the endoplasmic reticulum and in maintaining its function.

Keywords: Channel; STIM1; TRPV4; phosphorylation; protein-protein interaction.

Fig. (1)

Protein-protein interaction between STIM1 and the C-terminal domain of TRPV4. (A) Transmembrane topology of mouse TRPV4 (871 aa). The three ankyrin binding repeats (ANK; gray circles), the six trans-membrane regions (TM1_TM6), the STIM1 binding site (STIM1), and the putative SGK1 phosphorylation site (serine824) are indicated. The C-terminal cytoplasmic region of TRPV4(Δ718-871) is also indicated. The putative SGK1 phosphorylation site (serine 824) is present in the STIM1 domain (812-832 aa) of the TRPV4 channel. The line indicates the GST C-terminal cytoplasmic region of TRPV4(Δ718-871) or mutant (S824A) fusion protein, the TRPV4 mutant site (S824A or S824D) is compared with the wild-type (WT; Gene Bank no. BC127052). (B) Alignment of TRPV4 WT, S824A, and S824D with the consensus SGK1 substrate motif. The putative SGK1 phosphorylation site (serine 824) of TRPV4 is located in the specific conserved SGK1 substrate [R-X-R-X-X-(S/T)ϕ]. S824A and S824D were constructed using site-directed mutagenesis. (C) Interaction between TRPV4 and STIM1 in MDCK cells. Following immunoprecipitation (IP) using an anti-TRPV4 antibody, immunoblot (IB) analysis was performed using an antibody against STIM1 (left). Conversely, STIM1 immunoprecipitated complexes were subjected to immunoblot analysis using an anti-TRPV4 antibody (right). Co-immunoprecipitation of STIM1 with TRPV4 confirmed the formation of a TRPV4-STIM1 complex. The negative control for immunoprecipitation was an unrelated antibody. The control for western blot analysis was an antibody against actin (bottom). (D) MDCK cells were examined by direct immunofluorescence microscopy. The figures show TRPV4 (green), STIM1 (red), and merged (yellow) confocal microscopic images. (E) The C-terminal of TRPV4 is required for interaction with STIM1. After incubation of GST-TRPV4WT or the deletion mutant fusion protein (lacking aa 718-871) with MDCK cell lysate, the purified GST bead was immunoblotted with an antibody against STIM1 (upper lane). The deletion mutant lacking the C-terminal (aa 718-871) did not pull down STIM1 (right lane), whereas GST TRPV4 WT did (left lane).

Fig. (2)

The effect of phosphorylation of TRPV4 serine 824 on the interaction between TRPV4 and STIM1. (A) Co-immunoprecipitation of TRPV4 WT, S824A, or S824D with STIM1. HEK 293 cells were transiently transfected with EGFP-TRPV4 WT or S824A plasmid. After 48 h the cells were lysated, and pull-down of total protein was conducted with protein Aagarose beads. Western blot assays were performed with rabbit anti-TRPV4 or anti-STIM1 antibody. (B) Pull-down analysis of STIM1 with GST fusion C-terminal TRPV4 WT, S824A, or S824D. GST-fusion proteins encompassing C-terminal TRPV4 domains were constructed and expressed in E. coli. Approximately 2 mg of WT, S824A or S824D fusion protein bound to the glutathione-Sepharose bead were incubated with HEK 293 cell lysates.

Fig. (3)

Confocal microscopic images of cells transfected with EGFP-TRPV4 WT, S824A,or S824D. Cells were examined by direct immunofluorescence microscopy. The figures show EGFP-TRPV4 WT or mutant (S824A or S824D) (green), pmCherry-N1 STIM1 (red), and merged (yellow) confocal microscopic images. EGFP-TRPV4 WT or S824A showed maximal colocalization with pmCherry-N1 STIM1 in the cytosol (upper and middle lane). However, EGFP-TRPV4 S824D was principally detected in the focal adhesions, and was minimal colocalized with STIM1 at the Golgi apparatus (down lane).

Fig. (4)

Plasma membrane localization of TRPV4 WT, S824A, or S824D in cells after treatment with GSK650394 or insulin. (A) Confocal microscopic analysis of the transfected EGFP-TRPV4 S824A, WT, or S824D (green) after treatment with SGK1 inhibitor (GSK 650397, left), activator (insulin, right) for 12 h, and the serum free control (middle). The localization of TRPV4 WT (which can be phosphorylated on serine824 by SGK1) was altered by treatment with GSK650394 or insulin. The image is representative of five repeat experiments. (B) Effects of 4-αPDD on intracellular calcium concentration change [Ca²⁺]_i of TRPV4 WT, S824A, or S824D, expressed by the absorption at 488 nm of argon-ion laser in HEK 293 cells (as an arbitrary % unit).The average the maximum calcium flux represents below the graph (n=5).

Fig. (5)

Putative model for the regulation of the interaction between TRPV4 and STIM1 by phosphorylation on TRPV4 serine 824. TRPV4 WT can be activated by the interaction with proteins such as STIM1 through association/dissociation from its C-terminal cytoplasmic domain. The mechanism determining density of TRPV4 in the plasma membrane seems to be similar to that of other channels such as GLUT4 or AQP2. In contrast to TRPV4 S824A, S824D mutant cannot associate with STIM1 (for more detail, see the Discussion section).