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
View: Text | PDF
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

×

Figure 4

Role of WNK1 kinase domain in regulation of ROMK1 and interaction with ITSN.

Options: View larger image (or click on image) Download as PowerPoint
Role of WNK1 kinase domain in regulation of ROMK1 and interaction with I...
(A) Role of WNK1120–220 in WNK11–119 inhibition of ROMK1. *P < 0.05. (B–D) Effects of K233 and/or D368 mutations of WNK11–491 on WNK1 kinase activity (B), on ROMK1 current density (C), and on the interaction with ITSN SH3C (D). WNK11–491 carrying K233D and D368K single mutations are denoted K233D and D368, respectively. Double WNK11–491 mutant K233D/D368K is denoted KDDK. (B) Wild-type and mutant WNK11–491 were immunoprecipitated from lysates of transfected cells and used to phosphorylate purified kinase-dead GST-OSR1. Kinase-dead OSR1 was used here to prevent its autophosphorylation (37, 38). Autophosphorylation of WNK11–491 is denoted as WNK1. (C) Cells were transfected with ROMK1 plus wild-type WNK11–491 or one of the mutants. *P < 0.05 versus ROMK1 alone. NS, not significantly different versus ROMK1 alone. (D) GST-ITSN-SH3C was used to pull down wild-type WNK11–491 or mutants as described in Figure 3D.