TY - JOUR
T1 - Multistep regulation of autophagy by WNK1
AU - Kankanamalage, Sachith Gallolu
AU - Lee, A. Young
AU - Wichaidit, Chonlarat
AU - Lorente-Rodriguez, Andres
AU - Shah, Akansha M.
AU - Stippec, Steve
AU - Whitehurst, Angelique W.
AU - Cobb, Melanie H.
N1 - Funding Information:
We thank members of the M.H.C laboratory and former member Hashem Dbouk, as well as Joseph Albanesi (Department of Pharmacology, University of Texas Southwestern) and Kate Luby-Phelps (Department of Cell Biology and Live Cell Imaging Facility, University of Texas Southwestern) for their advice about this project. The work was supported by NIH Grant R01GM53032 (to M.H.C), Welch Foundation Grant I1243 (to M.H.C), and National Cancer Institute Grant P30CA142543 to the Harold C Simmons Comprehensive Cancer Center (Live Cell Imaging Facility). S.G.K. was supported in part by the Cancer Intervention and Prevention Discoveries Program RP140110.
PY - 2016/12/13
Y1 - 2016/12/13
N2 - The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phospha-tidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phos-phorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related pro-line-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or-starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.
AB - The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phospha-tidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51-like kinase 1 (ULK1), its phos-phorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related pro-line-alanine-rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or-starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.
KW - PI3KC3
KW - SPAK
KW - ULK1
KW - UVRAG
KW - Vps34
UR - http://www.scopus.com/inward/record.url?scp=85005999176&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85005999176&partnerID=8YFLogxK
U2 - 10.1073/pnas.1617649113
DO - 10.1073/pnas.1617649113
M3 - Article
C2 - 27911840
AN - SCOPUS:85005999176
SN - 0027-8424
VL - 113
SP - 14342
EP - 14347
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 50
ER -