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.
Highlights
Many important downstream effectors of phosphatidylinositol 3kinase (PI3K) are protein kinases
We have shown here that Serum- and glucocorticoid-regulated kinase 3 (Sgk3) exhibits negligible activity in the absence of activation loop phosphorylation and that PI3P significantly increases the basal activity of prephosphorylated Sgk3
Like other AGC kinases regulated by phospholipids, including Akt and protein kinase C (PKC), the PI3P-binding pocket of the phox homology (PX) domain of Sgk3 is sequestered in an autoinhibitory, intramolecular conformation
Summary
Many important downstream effectors of PI3K are protein kinases. Protein phosphorylation is a ubiquitous post-translational modification that regulates cell signaling and is carried out by the arsenal of more than 500 protein kinases in the human proteome [5]. A third site, called the turn motif, in the tail, regulates the stability of Akt and protein kinase C (PKC) [12, 13], but its phosphorylation has not been reported in Sgk3. Sgk3 is recruited to endosomes via its PI3P-binding phox homology (PX) domain [15, 16], which promotes phosphorylation of its activation loop and hydrophobic motif by PDK1 and mTORC2, respectively [9, 15, 17].
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