In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate

dc.contributor.authorPokorny, Daniel
dc.contributor.authorTruebestein, Linda
dc.contributor.authorFleming, Kaelin D.
dc.contributor.authorBurke, John E.
dc.contributor.authorLeonard, Thomas A.
dc.date.accessioned2021-07-12T16:58:09Z
dc.date.available2021-07-12T16:58:09Z
dc.date.copyright2021en_US
dc.date.issued2021
dc.description.abstractSerum- 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.en_US
dc.description.reviewstatusRevieweden_US
dc.description.scholarlevelFacultyen_US
dc.description.sponsorshipThis work was supported by the Austrian Science Fund (FWF) grants P28135, P30584 and P33066 to T.L. L.T. was supported by a Hertha Firnberg Postdoctoral Fellowship from the Austrian Science Fund (FWF T915). J.E.B. is supported by a Michael Smith Foundation for Health Research (MSFHR) Scholar award (17686), and an operating grant from the Cancer Research Society (CRS-24368).en_US
dc.identifier.citationPokorny, 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.en_US
dc.identifier.urihttps://doi.org/10.1016/j.jbc.2021.100919
dc.identifier.urihttp://hdl.handle.net/1828/13104
dc.language.isoenen_US
dc.publisherJournal of Biological Chemistryen_US
dc.rightsAttribution 2.5 Canada*
dc.rights.urihttp://creativecommons.org/licenses/by/2.5/ca/*
dc.subjectphospholipiden_US
dc.subjectserine/threonine protein kinaseen_US
dc.subjectendosomeen_US
dc.subjectphosphatidylinositide 3-kinase (PI 3-kinase)en_US
dc.subjectallosteric regulationen_US
dc.subjecthydrogen-deuterium exchange mass spectrometry (HDX-MS)en_US
dc.subjectSgk3en_US
dc.subjectCISKen_US
dc.titleIn vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphateen_US
dc.typePostprinten_US

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