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 2

ITSN is important for long WNK1 regulation of ROMK1.

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ITSN is important for long WNK1 regulation of ROMK1. (A) Domain structur...
(A) Domain structure of ITSN1s. It contains 2 EH domains, a central coiled-coil (CC) region, and 5 consecutive SH3 domains. The EH domains anchor ITSN to clathrin coat. The SH3 domains recruit dynamin, synaptojanin, and other endocytic proteins. (B) Amino acids 1–491 of long WNK1 interacted specifically with the ITSN SH3 C domain. GST fusion proteins were used to pull down myc-tagged WNK11–491 from transfected cell lysates. Western blot analysis was performed with anti-GST and anti-myc antibodies, respectively. Amphi, amphiphysin; Endo, endophilin. (C) Evidence for knockdown of endogenous ITSN1. Endogenous ITSN1s was detected by an anti-ITSN antibody. Western blot analysis of lysates from mock-transfected (control) cells and cells transfected with HA-tagged ITSN1s probed by anti-ITSN and anti-HA antibodies is shown on the right. (D) Knockdown of ITSN1 increased surface abundance of ROMK1. (E) Knockdown of ITSN1 increased baseline whole-cell ROMK1 current density and prevented the decrease caused by WNK11–491. Cells were transfected with ROMK1 plus indicated constructs. (F) Overexpression of dominant-negative ITSN1 increased baseline whole-cell ROMK1 current density and prevented the decrease caused by WNK11–491. *P < 0.05 versus ROMK1 alone.