NRBP1 pseudokinase binds to and activates the WNK pathway in response to osmotic stress- Raw Data for Supplementary Figures
-
1.
MRC Protein Phosphorylation and Ubiquitylation Unit
-
2.
University of Dundee
Description
WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif binding conserved C-terminal (CCT) domain, similar to the CCT domains in WNKs, SPAK and OXSR1. Knockdown or knock-out of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modelling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicates NRBP1 and likely its close homologue NRBP2, functions as an upstream activator of the WNK pathway.
|
Figure |
No |
Blots (WCL-Whole cell lysates) IP-Immunoprecipitation |
|
SFig 1A |
1 |
WCL probed with pSPAK S371 and GAPDH antibody |
|
|
2 |
WCL full membrane probed with Streptavidin HRP antibody |
|
|
3 |
WCL probed with GFP antibody to detect GFP-WNK1 |
|
SFig 1D |
1 |
Streptavidin pulldown probed for WNK1, NRBP1 and ELOB |
|
|
2 |
Streptavidin pulldown probed for TSC22D4 and TSC22D2 |
|
|
|
|
|
SFig 3B |
1 |
WCL (upper) and GFP IP (lower) probed with HA antibody to detect HA-NRBP1 |
|
|
2 |
WCL (upper) and GFP IP (lower) probed with GFP antibody to detect GFP-TSC22D4 and empty GFP |
|
|
|
|
|
SFig 4B |
1 |
Blotting with pNRBP1 T232(low exposure), GAPDH and TSC22D4 antibody |
|
|
2 |
pNRBP1 T232b reprobed with GFP antibody to detect total GFP-NRBP1 levels |
|
|
3 |
Blotting with TSC22D2, pNRBP1 T232(high exposure) and SPAK antibody |
|
SFig 4C |
1 |
Blotting for GST |
|
|
2 |
Blotting for pNRBP1 T232 |
|
|
3 |
Blotting for NRBP1 |
|
|
|
|
|
SFig 5A |
1 |
GFP IP blot probed with GFP antibody for GFP-NRBP1 and TSC22D4 antibody WCL probed with GFP antibody for GFP-NRBP1 |
|
|
2 |
WCL probed for pSPAK S371 and TSC22D2 antibody |
|
|
3 |
WCL probed for WNK1 |
|
|
4 |
WCL probed for SPAK |
|
|
5 |
WCL probed for GAPDH |
|
|
|
|
|
SFig 7C |
1 |
WCL probed with SPAK antibody |
|
|
2 |
NRBP1 IP blot probed with NRBP1 (sigma antibody) |
|
|
3 |
WCL probed for TSC22D4 |
|
|
4 |
WCL probed for TSC22D2 and GAPDH |
|
|
5 |
WCL probed for pSPAK S371 |
|
|
|
|
|
SFig 8C |
1 |
Blotting with HA antibody to detect HA tagged NRBP1 & NRBP2 in WCL and HA & GFP IP |
|
|
2 |
Blotting with GFP antibody to detect GFP tagged NRBP1 & NRBP2 in WCL and HA & GFP IP |
|
SFig 8D |
1 |
HA IP blot probed with WNK1 and GFP antibody to detect endogenous WNK1 and GFP-WNK1 respectively. |
|
|
2 |
WCL probed with WNK1 and GFP antibody to detect endogenous WNK1 and GFP-WNK1 respectively. |
|
|
3 |
HA IP blot probed with NRBP1 |
|
|
4 |
HA IP blot probed with TSC22D4 |
|
|
5 |
WCL probed with pSPAK S371, SPAK, TSC22D4 and GFP antibody |
|
|
6 |
HA IP blot probed with GFP antibody |
|
|
|
|
|
SFig 8E |
1 |
GFP IP (above) and WCL (below) probed with flag antibody to detect Flag TSC22D4 |
|
|
2 |
GFP IP probed with GFP antibody to detect GFP CCTs and GFP empty |
|
|
3 |
WCL probed with GFP antibody to detect GFP CCTs |
|
SFig 8F |
1 |
GFP IP (above) and WCL (below) probed with Flag antibody to detect Flag TSC22D2 |
|
|
2 |
GFP IP probed with GFP antibody to detect GFP CCTs and GFP empty |
|
|
3 |
WCL probed with GFP antibody to detect GFP CCTs |
|
|
|
|
|
SFig 9 |
1 |
Blotted with NRBP1 antibody |
|
|
2 |
Blotted with NRBP1 (left) and NRBP2 (right) antibody with HA-NRBP1 and HA-NRBP2 overexpression. |
|
|
3 |
Blotted with NRBP1 (yellow) and NRBP2 (red) antibody |
|
|
4 |
Blotted with TSC22D4 (right) and TSC22D2 (left) antibody |
|
|
|
|
Files
SFig 1A 1.jpg
Files
(67.3 MB)
| Name | Size | Download all |
|---|---|---|
|
md5:da931fb57f433294adce54f1205ee442
|
50.4 kB | Preview Download |
|
md5:f690da58f271e6e200e114f6eb49edcc
|
3.8 MB | Preview Download |
|
md5:aa5a34e39259af14d77607ba23c8f68f
|
35.3 kB | Preview Download |
|
md5:b1bac53b488973755d7f94368d637790
|
6.6 MB | Preview Download |
|
md5:27d4c2f0c112b259583c948941528457
|
4.0 MB | Preview Download |
|
md5:914b104b409a90cd41724b960e368cc4
|
259.5 kB | Preview Download |
|
md5:61630f325cf861a13a5560e7de11f619
|
12.8 MB | Preview Download |
|
md5:e237fa6a6702a49b9d6eaeca58cd27e2
|
139.8 kB | Preview Download |
|
md5:446999e13b32b13c3bb5e9a623f6e845
|
36.1 kB | Preview Download |
|
md5:e2a86a92f2850850b66bb359a3622ab9
|
1.1 MB | Preview Download |
|
md5:5540e978a9b28c451719fcd9e7daeecd
|
585.3 kB | Preview Download |
|
md5:61d66076486c301d4a9ba7afeccaaf98
|
877.6 kB | Preview Download |
|
md5:07d0f4bcc5d485501e5f3a2b68f7860f
|
2.7 MB | Preview Download |
|
md5:76b3847b107d1656272ceedc171c8194
|
171.8 kB | Preview Download |
|
md5:99003de46a604b47a85bc210916afc35
|
70.6 kB | Preview Download |
|
md5:3fd404d13d1a6883b2aefcf96ed89405
|
57.0 kB | Preview Download |
|
md5:d62def04e3f03793465577827f5375ff
|
60.9 kB | Preview Download |
|
md5:d13bdb25ebe2c7d7d7479562cff4cb95
|
33.6 kB | Preview Download |
|
md5:c9794f299b3b3f97d3fc0733329466dc
|
74.0 kB | Preview Download |
|
md5:e0e14d94b1f97516e28c05a09acdc5ab
|
154.1 kB | Preview Download |
|
md5:b1bbcd208131a7be34063ca015607ca1
|
57.4 kB | Preview Download |
|
md5:710cb7b9f375e1fbf97879e3199c6060
|
86.7 kB | Preview Download |
|
md5:77b7b5c160cc3330ec9af49475837d34
|
74.2 kB | Preview Download |
|
md5:fa8117e5d4cfd001f968707d875aeed7
|
2.8 MB | Preview Download |
|
md5:b6e78e0519d5587069f3ef5b71f3d950
|
153.4 kB | Preview Download |
|
md5:24d1cbcdac7b01bc67c48f00e20ef591
|
4.4 MB | Preview Download |
|
md5:a0f965f3a0c86a7afaa619516d8986a5
|
6.0 MB | Preview Download |
|
md5:afaf3fc785986f5547b784541eea1e40
|
195.7 kB | Preview Download |
|
md5:7ebf33163dc77ea6106e0e868221ee42
|
121.3 kB | Preview Download |
|
md5:23216523b9c0aba4f4387e337964fd0f
|
13.4 MB | Preview Download |
|
md5:1bf36f8029d4b2ac7f65843777d700e8
|
202.4 kB | Preview Download |
|
md5:3140405c9c0bd614842339fe01d01d42
|
951.2 kB | Preview Download |
|
md5:e062b93e599d1a63207ed02038083b50
|
340.6 kB | Preview Download |
|
md5:a6b10136980014c9fb1449a1104e8ffb
|
428.0 kB | Preview Download |
|
md5:1521b52c4262693a3590acfcdf79120c
|
814.0 kB | Preview Download |
|
md5:7dcfe57a855db9eebad1d82837608525
|
341.2 kB | Preview Download |
|
md5:f9baa473dfd0a582280df0489a59446b
|
438.4 kB | Preview Download |
|
md5:464a9aad7c981a0751d7eb385827a117
|
815.8 kB | Preview Download |
|
md5:fff2c1635e329edf69d44552bcea1ec0
|
567.6 kB | Preview Download |
|
md5:b32773a174f0ff8d53d850376a1e0154
|
934.9 kB | Preview Download |
|
md5:833941ba97e9263b71a1820ade02ea65
|
505.5 kB | Preview Download |
Additional details
Dates
- Submitted
-
2025-04