Intersectin links WNK kinases to endocytosis of ROMK1
Guocheng He, … , Shao-Kuei Huang, Chou-Long Huang
Guocheng He, … , Shao-Kuei Huang, Chou-Long Huang
Published April 2, 2007
Citation Information: J Clin Invest. 2007;117(4):1078-1087. https://doi.org/10.1172/JCI30087.
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Research Article Nephrology

Intersectin links WNK kinases to endocytosis of ROMK1

Abstract

With-no-lysine (WNK) kinases are a novel family of protein kinases characterized by an atypical placement of the catalytic lysine. Mutations of 2 family members, WNK1 and WNK4, cause pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. WNK1 and WNK4 stimulate clathrin-dependent endocytosis of renal outer medullar potassium 1 (ROMK1), and PHA2-causing mutations of WNK4 increase the endocytosis. How WNKs stimulate endocytosis of ROMK1 and how mutations of WNK4 increase the endocytosis are unknown. Intersectin (ITSN) is a multimodular endocytic scaffold protein. Here we show that WNK1 and WNK4 interacted with ITSN and that the interactions were crucial for stimulation of endocytosis of ROMK1 by WNKs. The stimulation of endocytosis of ROMK1 by WNK1 and WNK4 required specific proline-rich motifs of WNKs, but did not require their kinase activity. WNK4 interacted with ROMK1 as well as with ITSN. Disease-causing WNK4 mutations enhanced interactions of WNK4 with ITSN and ROMK1, leading to increased endocytosis of ROMK1. These results provide a molecular mechanism for stimulation of endocytosis of ROMK1 by WNK kinases.

Authors

Guocheng He, Hao-Ran Wang, Shao-Kuei Huang, Chou-Long Huang

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Figure 6

Representative experiments showing the effects of PHA2 mutation on WNK4 regulation of ROMK1 and interaction with ITSN.

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Representative experiments showing the effects of PHA2 mutation on WNK4 ...
(A) Amino acids 545–561 of WNK4 contain 3 PXXP motifs and disease-causing mutations. Double mutations of P548A/P555A disrupted all 3 PXXP motifs (denoted PA2). (B) Triple PXXP mutations prevented the decrease of surface ROMK1 caused by WNK41–584. (C) ITSN did not interact with triple PXXP mutations of WNK41–584. GST fusion proteins were used to pull down myc-tagged WT WNK41–584 or WNK41–584 with the triple PXXP mutation from lysates of transfected cells. (D) Disease-causing mutations increased the ability of WNK4 to decrease surface ROMK1 levels. (E) Effects of PXXP motifs and disease-causing mutations of WNK4 on ROMK1 currents. Cells were transfected with ROMK1 plus indicated constructs. *P < 0.05 versus ROMK1 alone. #P < 0.05 versus ROMK1 plus WNK41–584. (F) SH3A–SH3E interacted with WT WNK41–584 and the E559K mutant. H6-tagged WNK41–584 protein was used to pull down HA-tagged ITSN SH3A–SH3E from lysates of transfected cells. The amount of ITSN SH3 shown for supernatant (S) and pellet (P) represent 2% and 50% of their totals, respectively. To compare the efficacy of ITSN SH3 domain pulldown by WT WNK41–584 and E559K, the intensity of ITSN SH3 band in pellet was quantified and normalized to that in supernatant (WNK41–584, 0.32; WNK41–584 plus E559K, 0.83). This is in spite of that the intensity of E559K mutant (used for pulldown) was 65% that of WT (mean ± SEM, WNK41–584, 0.38 ± 0.15; WNK41–584 plus E559K, 0.81 ± 0.21; P < 0.05; n = 4 per group).