Activation of PI3-Kinase Stimulates Endocytosis of ROMK via Akt1/SGK1-Dependent Phosphorylation of WNK1

Abstract

WNK kinases stimulate endocytosis of ROMK channels to regulate renal Kϩ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulininduced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominantnegative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1.

Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action

“…Therefore, the pattern of WNK1 gene expression in the distal nephron positions WNK1 downstream of SGK1, with NEDD4-2 being an intermediary that suppresses WNK1 protein abundance in the absence of aldosterone. Previous observations that SGK1 directly phosphorylates and activates L-WNK1 through interactions with its N-terminus further support the notion that WNK1 functions downstream of SGK1 in the ASDN (43). Thus, during high aldosterone states, NEDD4-2 inhibition would be expected to increase WNK1 protein abundance, expanding the supply of L-WNK1 kinases that can be activated by SGK1 and trigger downstream signaling through SPAK and OSR1 (Figure 10).…”

“…WNK4 abundance was also significantly lower in these mice, possibly due to known direct interactions between SGK1, WNK1, and WNK4 (42)(43)(44). Consistent with decreased WNK1 and WNK4 kinase activity, downstream SPAK/OSR1 phosphorylation was also reduced (Figure 9, A and B).…”

Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

“…However, mutation of T60 inhibits SGK activation by WNKs, suggesting that phosphorylation of T60 is a permissive event that exerts positive feedback on some WNK1 functions (24,36). Phosphorylation of T60 is required for maximal activation of ENaC by WNK1, consistent with the effect on SGK1 activation (36); regulation of the renal outer medullary potassium channel by WNK1 also depends on phosphorylation of T60 (56). WNK1 activity is essential for phospholipase C beta PLCβ function downstream of Gq-coupled receptors, which may also involve PI3K (57).…”

LINGO-1 Receptor Promotes Neuronal Apoptosis by Inhibiting WNK3 Kinase Activity

“…As a serine/threonine kinase, WNK1 has been reported to be a substrate for Akt-mediated phosphorylation (35) and is dependent on PI3K/Akt-mediated endocytosis of ROMK (36). Recent studies suggest that WNK3 can also be phosphorylated by Akt (37).…”