Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1−/− ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1L155E/L155E knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.
- Gordon's syndrome
- mass spectrometry (MS)
- 3-phosphoinositide-dependent protein kinase-1 (PDK1)
- phosphoinositide 3-kinase (PI 3-kinase)
- pseudohypoaldosteronism type II (PHAII)
Abbreviations used: PKA, cAMP-dependent protein kinase; PKB, protein kinase B; PKC, protein kinase C; AGC kinase, similar to PKA, protein kinase G and PKC protein kinase; ERK, extracellular-signal-regulated protein kinase; ES, embryonic stem cells; GSK3, glycogen synthase kinase-3; GST, glutathione S-transferase; IGF1, insulin-like growth factor; KD, kinase dead; MBP, myelin basic protein; MSK1, mitogen- and stress-activated protein kinase-1; PDK1, 3-phosphoinositide-dependent protein kinase-1; PHAII, pseudohypoaldosteronism type II; PI 3-kinase, phosphoinositide 3-kinase; RSK, p90 ribosomal S6 kinase; S6K, p70 ribosomal S6 kinase; SGK, serum- and glucocorticoid-induced protein kinase; WNK1, with no K (lysine) protein kinase-1; CREB, cAMP-response-element-binding protein; BAD, Bcl-2/Bcl-XL-antagonist, causing cell death; GFP, green fluorescent protein.
- The Biochemical Society, London ©2004