Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

Abstract

The serum-and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is upregulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including 4 endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are poorly phosphorylated by Akt as they possess an n+1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with 14H inhibitor. SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.

Design and Characterization of SGK3-PROTAC1, an Isoform Specific SGK3 Kinase PROTAC Degrader

“…44 Furthermore, it would be interesting to explore the effects that SGK3 PROTAC degraders have on selective SGK3 substrates such as STX7 and STX12 that have recently been described that are not phosphorylated by Akt isoforms. 46 The finding that growth of SGK3 dependent cancer cell lines is suppressed more efficiently by SGK3-PROTAC1 than achieved by the 14H non-PROTAC inhibitor provides a further example of the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. Other examples include the recent finding that a BCR-ABL degrader displays more sustained inhibition of chronic myelogenous leukemia cell growth than can be achieved by a conventional ABL kinase inhibitor.…”

“…Measurement of the stability of the ternary complexes of SGK3 PROTAC compounds with SGK/S6K isoforms and the VHL E3 ligase would be important to better understand the potency and specificity of these PROTACs . Furthermore, it would be interesting to explore the effects that SGK3 PROTAC degraders have on selective SGK3 substrates such as STX7 and STX12 that have recently been described that are not phosphorylated by Akt isoforms . The finding that growth of SGK3 dependent cancer cell lines is suppressed more efficiently by SGK3-PROTAC1 than achieved by the 14H non-PROTAC inhibitor provides a further example of the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors.…”

In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate

“…Notwithstanding the similarity in their kinase domains, it is clear that their activity profiles would overlap in such a model, since the PI(3,4)P2 that drives PDK1 and Akt activation must be present in membranes that support PI3P-and PDK1-mediated Sgk3 activation. Indeed, a recent phosphoproteomic study identified several Sgk3specific substrates localized on endosomes (59). The reported role of Sgk3 in driving human cancers in the absence of Akt hyperactivation is then also reconcilable with this model and reinforces the potential value of Sgk3 as a therapeutic target either as a monotherapy or in combination with Akt inhibitors.…”