Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan;32(1):208-219.
doi: 10.1096/fj.201700309R. Epub 2017 Sep 6.

Proline-dependent and Basophilic Kinases Phosphorylate Human TRPC6 at Serine 14 to Control Channel Activity Through Increased Membrane Expression

Affiliations
Free PMC article

Proline-dependent and Basophilic Kinases Phosphorylate Human TRPC6 at Serine 14 to Control Channel Activity Through Increased Membrane Expression

Henning Hagmann et al. FASEB J. .
Free PMC article

Abstract

Signaling via the transient receptor potential (TRP) ion channel C6 plays a pivotal role in hereditary and sporadic glomerular kidney disease. Several studies have identified gain-of-function mutations of TRPC6 and report induced expression and enhanced channel activity of TRPC6 in association with glomerular diseases. Interfering with TRPC6 activity may open novel therapeutic pathways. TRPC6 channel activity is controlled by protein expression and stability as well as intracellular trafficking. Identification of regulatory phosphorylation sites in TRPC6 and corresponding protein kinases is essential to understand the regulation of TRPC6 activity and may result in future therapeutic strategies. In this study, an unbiased phosphoproteomic screen of human TRPC6 identified several novel serine phosphorylation sites. The phosphorylation site at serine 14 of TRPC6 is embedded in a basophilic kinase motif that is highly conserved across species. We confirmed serine 14 as a target of MAPKs and proline-directed kinases like cyclin-dependent kinase 5 (Cdk5) in cell-based as well as in vitro kinase assays and quantitative phosphoproteomic analysis of TRPC6. Phosphorylation of TRPC6 at serine 14 enhances channel conductance by boosting membrane expression of TRPC6, whereas protein stability and multimerization of TRPC6 are not altered, making serine 14 phosphorylation a potential drug target to interfere with TRPC6 channel activity.-Hagmann, H., Mangold, N., Rinschen, M. M., Koenig, T., Kunzelmann, K., Schermer, B., Benzing, T., Brinkkoetter, P. T. Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.

Keywords: MAPK; cyclin-dependent kinase 5; glomerular disease; podocyte; proteinuria.

Figures

Figure 1.
Figure 1.
Identification of novel serine phosphosites in TRPC6-MAPK and Cdk5 phosphorylate TRPC6 at serine residues. A) Human TRPC6 was transiently expressed in HEK 293T cells. After purification of phosphopeptides from crude cell lysates, liquid chromatographic tandem mass spectrometry analysis identified novel phosphorylation sites. Serine residues in positions 4, 13/14, and 814 (red asterisks) were identified as phosphorylation targets. Schematic shows TRPC6 and phosphopeptides (blue). Transmembrane domains and intra- or extracellular loops are shown in gray. B, C) Mass-to-charge ratio spectra of serine 4 and 13/14 phosphorylation sites. Green, peaks with phosphorylation loss; red, b ions; blue, y ions. D) HEK 293T cells were transiently transfected as indicated. After immunoprecipitation of V5.TRPC6, immunoblot analysis was performed with phospho-serine-specific motif antibody, showing serine phosphorylation of TRPC6 in presence of ERK, p38, and Cdk5/p35. Restaining was performed with V5-specific antibody.
Figure 2.
Figure 2.
Cdk5/p35 phosphorylates TRPC6 at serine 14. A) HEK 293T cells were transiently transfected as indicated. After immunoprecipitation for V5.TRPC6, immunoblot analysis was performed with phospho-serine-specific motif antibody, showing TRPC6 serine phosphorylation only in presence of WT Cdk5/p35. B) Densitometry of phospho-serine motif immunoblot normalized to Cdk5 WT transfected cells detects significantly more phosphorylation in Cdk5/p35 transfected cells compared to Cdk5-dn/p35 and Cdk5 WT alone (n = 3; Student’s t test). #P > 0.05 (not significant); *P < 0.05. C) TRPC6 WT and phospho-ablating mutants of TRPC6 were analyzed in radioactive in vitro kinase assay. TRPC6 WT (WT), TRPC6 S4A, or TRPC6 S14A (all V5 tagged) were expressed in HEK 293T cells and precipitated from cell lysates. After in vitro addition of recombinant Cdk5/p35 and 32P-labeled ATP to immunoprecipitates of TRPC6, phosphorylation of TRPC6 was detected by autoradiography after SDS-PAGE. TRPC6 phosphorylation is abrogated when serine 14 is replaced by alanine. Coomassie blue–stained gel served as loading control.
Figure 3.
Figure 3.
TRPC6 phosphorylation site at serine 14 is conserved across species. A) TRPC6 protein sequence was analyzed by CLUSTAL-O algorithm. Sequence analysis and alignment of TRPC6 reveals high conservation of domain around serine 14 across species. Proline at +1 position is not conserved in mice. B) HEK 293T cells expressing human or mouse TRPC6 and active Cdk5/p35 or Cdk5-dn/p35 were subjected to quantitative phosphoproteomics. Abundance of phospho-S14 TRPC6 is plotted for cells expressing active Cdk5/p35 compared to dominant-negative Cdk5/p35 (n = 3; Student’s t test). #P > 0.05 (not significant); **P < 0.01.
Figure 4.
Figure 4.
Phosphorylation of TRPC6 at serine 14 modulates channel activity. TRPC6 channel activity was assessed in double-electrode voltage-clamp experiments in Xenopus oocytes after stimulation with 10 µM OAG. Coexpression of Cdk5 WT and p35 leads to enhanced conductance of TRPC6 channel. A) Summary of whole-cell currents measured at clamp voltage Vc = −60 mV. TRPC6 induced inward current that was augmented in presence of Cdk5/p35, similar to coexpression with podocin. OAG had no effect on mock-injected oocytes. B) Summaries of effects of coexpression with phospho-deficient mutant TRPC6-S14A or Cdk5-dn. Data are presented as means ± sem; values in parentheses indicate number of experiments. *P < 0.05 vs. mock vector (A) or TRPC6 (B) injection (ANOVA); $P < 0.05 vs. TRPC6 (A) or TRPC6 + Cdk5 + p35 (B) (ANOVA).
Figure 5.
Figure 5.
Phosphorylation at serine 14 does not alter protein stability or oligomerization characteristics of TRPC6. A) HEK 293T cells were transiently transfected with V5-tagged constructs as indicated and treated with 25 µg/ml cycloheximide for time indicated. Band density was quantified after immunoblot analysis of cell lysates with V5-specific antibody and normalized to untreated control. No significant difference in protein stability was detected for TRPC6 WT and phospho mutants (n = 3; Student’s t test). B) Multimerization of TRPC6 was assessed in coimmunoprecipitation experiments. FLAG- and V5-tagged TRPC6 cDNA constructs were transiently expressed in HEK 293T cells as indicated. After immunoprecipitation with FLAG-specific antibody, immunoblotting was performed with V5-specific antibody and restaining of precipitates with FLAG-specific antibody. C) Densitometry quantification of V5 immunoblot shows no significant difference in multimerization for TRPC6 WT and phospho mutants (AU, arbitrary units; n = 3; Student’s t test). D) To investigate complex size of native TRPC6 multimers, HEK 293T cells were transiently transfected with V5-tagged TRPC6 cDNA constructs as indicated, and cell lysates were subjected to velocity gradient centrifugation. Analysis of fractions on immunoblot using V5-specific antibody shows similar sedimentation pattern of TRPC6 WT and TRPC6 S14A and S14D (n = 3). #P > 0.05 (not significant).
Figure 6.
Figure 6.
Phosphorylation of TRPC6 at serine 14 affects surface expression of TRPC6. A) HEK 293T cells were transiently transfected as indicated. After surface biotinylation, cell lysis and streptavidin precipitation immunoblot analysis were performed using V5-specific antibody. B) Densitometry of V5 immunoblot of biotinylated precipitates detects significantly more TRPC6 protein for phospho-mimicking mutant S14D (AU, arbitrary units; n = 3; t test). #P > 0.05 (not significant); *P < 0.05, **P < 0.01. C) HeLa cells transiently expressing V5-tagged TRPC6 cDNA constructs as indicated were stained with V5-specific antibody. At equal exposure times, more prominent immunoreactivity for V5 was found in plasma membrane of cells expressing TRPC6 S14D (white arrowhead).
Figure 7.
Figure 7.
TRPC6 phosphorylation at serine 14 affects plasma membrane abundance in cultured human podocytes. A) Human podocytes transiently expressing V5-tagged TRPC6 cDNA constructs as indicated were stained with V5-specific antibody. Strong immunoreactivity for V5 is found in plasma membrane of cells expressing TRPC6 S14D (white arrowhead), whereas for TRPC6 S14A perinuclear accumulation is detected at equal exposure times. B) After hypertonic stress, V5.TRPC6 WT prominently localizes in plasma membrane of human podocytes. In contrast, V5.TRPC6 S14A reactivity is mostly restricted to endoplasmic reticulum. Treatment of V5.TRPC6 WT expressing human podocytes with MAPK/p38 inhibitor SB 203580 abrogates plasma membrane localization. Lysates separated in SDS-PAGE were analyzed by immunoblot with phospho-p38 and total-p38 specific antibody.

Similar articles

See all similar articles

Cited by 1 article

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback