The serine 814 of TRPC6 is phosphorylated under unstimulated conditions

Abstract

TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser(814) into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser(814) is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser(814)) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site.

Figure 1. MS/MS identification of potential phosphorylated residues on TRPC6.

A, Untreated T6.11 cells were lysed before TRPC6 was immunoprecipitated using an anti-HA antibody. The immunoprecipitated proteins were then deglycosylated with PNGaseF or not, before being separated by SDS-PAGE and stained with Colloidal Brilliant Blue. B, Sequence coverage of TRPC6 by nano-LC-MS/MS after tryptic digestion is highlighted. Ser⁸¹⁴ is shown in white on a black background. The transmembrane segments are underlined. Residues 931 to 939 represent the HA epitope. Total sequence coverage is 68%. C, Simplified LC-MS/MS spectrum of the FGISGpSHEDLSK peptide from TRPC6 phosphorylated at Ser⁸¹⁴. The phosphorylation of Ser⁸¹⁴ was confirmed by the mass assignments of fragmentation ions b5, b6, y6, and y7.

Figure 2. Metabolic labeling of TRPC6 and mutants reveals that Ser814 is phosphorylated.

A, Metabolic labeling was carried out as described in Experimental Procedures. HEK 293T cells transfected with wild-type or mutant TRPC6 were lysed and TRPC6 was immunoprecipitated using a mouse monoclonal anti-HA antibody (HAm) and separated by SDS-PAGE. The autoradiogram (upper panel) and the immunoblot using a rabbit polyclonal anti-HA antibody (HAr) (lower panel) are representative of five independent experiments. B, The histogram represents the relative amount of phosphorylated TRPC6 calculated as the ratio of phospho-TRPC6 (densitometric analysis of autoradiograms) to total TRPC6 (determined by scanning and quantifying the immunoblot signals) for each sample. The histogram is the average ± SD of five experiments. * P<0.03.

Figure 3. The S814A mutation does not alter TRPC6 activity.

A, [Ca²⁺]_i was recorded in HEK293T transiently transfected with pcDNA3 (open circles), TRPC6WT (open diamonds), or TRPC6^S814A (closed squares). The cells were incubated in the absence of extracellular Ca²⁺ (in the presence of 0.5 mM EGTA) for 30 s before being stimulated with 5 µM CCh. External Ca²⁺ (1.8 mM) was restored at 180 s. The graphs represent the average of 103 to 165 cells from one representative experiment. B, CCh-induced net Ca²⁺ entry (average of [Ca²⁺]_i values after 174–177 s subtracted from [Ca²⁺]_i values after 222–228 s) were calculated and graphed as averages ± SD of six independent experiments. * P<0.01.

Figure 4. The mutation of Ser⁸¹⁴ into Ala does not modify the level of expression nor the trafficking of TRPC6.

TRPC6 and TRPC^S814A were transfected in HEK293T cells. The cells were biotinylated with sulfo-NHS-SS-biotin, lysed, and incubated with streptavidin-agarose beads as described in Experimental Procedures. Proteins precipitated by streptavidin-agarose were separated by SDS-PAGE and detected with an anti-HA antibody (top). Aliquots of the cell lysates were collected before the incubation with streptavidin-agarose and analyzed directly by immunoblotting to determine the total amount of TRPC6 (middle) and actin (bottom) in the samples. Representative immunoblot of three independent experiments.

Figure 5. CK2 does not phosphorylate TRPC6.

A, T6.11 cells were labeled in the presence or not of 10 µM DMAT or 10 µM TBCA for 4 h before the cells were lysed and TRPC6 was immunoprecipitated and separated by SDS-PAGE. The autoradiogram is representative of six independent experiments. B, The densitometric analyses of autoradiograms and Western blots (not shown) from A and the calculated ratios are relative to the control (100%). The histogram represents the average ± SD of six independent experiments.

Figure 6. The inhibition of CK2 with specific inhibitors does not alter TRPC6 activity.

A, Fura-2-loaded T6.11 cells were left untreated (circles) or were treated for 4 h with 10 µM DMAT (squares) or 10 µM TBCA (triangles). CCh (5 µM) induced Ca²⁺ release in the absence of extracellular Ca²⁺ while Ca²⁺ restoration 2 min later induced Ca²⁺ entry. DMAT and TBCA concentrations were maintained during the assay. The graphs are representative of 176 to 227 cells from one typical experiment. B, Net maximal Ca²⁺ entries from A were calculated as in Figure 3 and are relative to the control (100%). The histogram represents the average ± SD of four independent experiments. C, The same protocol was carried out on A7r5 cells using 100 nM AVP instead of CCh. The net maximal Ca²⁺ entry was calculated relative to the control (100%). The histogram is the average ± SD of three independent experiments.