Intersectin links WNK kinases to endocytosis of ROMK1
Guocheng He, Hao-Ran Wang, Shao-Kuei Huang, Chou-Long Huang
Guocheng He, Hao-Ran Wang, Shao-Kuei Huang, Chou-Long Huang
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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 1

Effects of long WNK1 on ROMK1.

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Effects of long WNK1 on ROMK1. (A) Long WNK1 decreases surface abundance...
(A) Long WNK1 decreases surface abundance of GFP-ROMK1 via a dynamin-dependent mechanism. Surface abundance was measured by biotinylation assays. Equal expression of the total ROMK1 in transfected cells was confirmed by Western blot analysis. Expression of Myc-tagged full-length long WNK1 and dynamin-2 (WT or dominant-negative; DN) was examined by an anti-myc antibody. The molecular size of proteins is shown. (B) Knockdown of endogenous long WNK1 by siRNA (top) increased surface abundance of ROMK1 (bottom). HEK cells were transfected with GFP-ROMK1 alone. Control oligo, control oligonucleotides. (C) Validation of the integrity of biotinylation assay. Cells were transfected with GFP-ROMK1 and incubated with or without biotin before lysis. Biotinylated proteins were precipitated by streptavidin beads. HC, heavy chain. (D) Effects of long WNK1 on whole-cell and single ROMK1 channel activity. Top left: Current-voltage relationship of whole-cell ROMK1 currents. Top right: Long WNK1 decreased whole-cell ROMK1 currents. Bottom: Long WNK1 had no effects on single-channel conductance (at –100 mV) or open probability (Po) of ROMK1. Open probability was measured based on approximately 15 minutes recording from patches containing 1 active channel. *P < 0.05 vs ROMK alone. C, closed state; O, open state.