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.
Skip Nav Destination
Article navigation
Research Article|
February 15 2004
WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate
Alberto C. VITARI;
Alberto C. VITARI
1
*MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
1To whom correspondence should be addressed (e-mail a.c.vitari@dundee.ac.uk).
Search for other works by this author on:
Maria DEAK;
Maria DEAK
*MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Barry J. COLLINS;
Barry J. COLLINS
*MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Nick MORRICE;
Nick MORRICE
*MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Alan R. PRESCOTT;
Alan R. PRESCOTT
†Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Anne PHELAN;
Anne PHELAN
‡Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, U.K.
Search for other works by this author on:
Sian HUMPHREYS;
Sian HUMPHREYS
‡Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent CT13 9NJ, U.K.
Search for other works by this author on:
Dario R. ALESSI
Dario R. ALESSI
*MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
November 05 2003
Accepted:
November 11 2003
Accepted Manuscript online:
November 11 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 378 (1): 257–268.
Article history
Received:
November 05 2003
Accepted:
November 11 2003
Accepted Manuscript online:
November 11 2003
Citation
Alberto C. VITARI, Maria DEAK, Barry J. COLLINS, Nick MORRICE, Alan R. PRESCOTT, Anne PHELAN, Sian HUMPHREYS, Dario R. ALESSI; WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate. Biochem J 15 February 2004; 378 (1): 257–268. doi: https://doi.org/10.1042/bj20031692
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.