In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate
Date
2021
Authors
Pokorny, Daniel
Truebestein, Linda
Fleming, Kaelin D.
Burke, John E.
Leonard, Thomas A.
Journal Title
Journal ISSN
Volume Title
Publisher
Journal of Biological Chemistry
Abstract
Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein
kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P)
downstream of growth factor signaling via class I phosphatidylinositol 3-kinase
(PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on
endosomes. Upregulation of Sgk3 activity has recently been linked to a number of
human cancers; however, the precise mechanism of activation of Sgk3 is unknown.
Here, we use a wide range of cell biological, biochemical, and biophysical
techniques, including hydrogen-deuterium exchange mass spectrometry, to
investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is
regulated by a combination of phosphorylation and allosteric activation. We
demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX)
domain induces large conformational changes in Sgk3 associated with its activation,
and that the PI3P binding pocket of the PX domain of Sgk3 is sequestered in its
inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated
PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying
the mechanism of Sgk3 activation by PI3P, our findings open up potential
therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.
Description
Keywords
phospholipid, serine/threonine protein kinase, endosome, phosphatidylinositide 3-kinase (PI 3-kinase), allosteric regulation, hydrogen-deuterium exchange mass spectrometry (HDX-MS), Sgk3, CISK
Citation
Pokorny, D., Truebestein, L., Fleming, K. D., Burke, J. E., Leonard, T. A. (2021). In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate. Journal of Biological Chemistry.