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

Nazma Malik, Raja S. Nirujogi, Julien Peltier, Thomas Macartney, Melanie Wightman, Alan Prescott, Robert Gourlay, Matthias Trost, Dario Alessi (Lead / Corresponding author), Athanasios Karapetsas (Lead / Corresponding author)

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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 up-regulated 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 four 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 inefficiently 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 a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the 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.

Original languageEnglish
Pages (from-to)3081-3107
Number of pages27
JournalBiochemical Journal
Volume476
Issue number20
Early online date14 Oct 2019
DOIs
Publication statusPublished - 30 Oct 2019

Fingerprint

Qa-SNARE Proteins
Phosphorylation
Phosphotransferases
SNARE Proteins
Substrates
Phosphatidylinositol 3-Kinases
Protein Isoforms
Proteins
HEK293 Cells
Endosomes
Phosphatidylinositols
Biomarkers
Cell membranes
Neoplasms
Cell Membrane
Pharmacology
Breast Neoplasms
Tumors
Cells
Growth

Keywords

  • SGK3
  • Phosphoproteomics
  • phosphoinositide 3-kinase
  • Akt
  • syntaxins
  • phosphoproteomics

Cite this

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title = "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 up-regulated 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 four 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 inefficiently 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 a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the 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.",
keywords = "SGK3, Phosphoproteomics, phosphoinositide 3-kinase, Akt, syntaxins, phosphoproteomics",
author = "Nazma Malik and Nirujogi, {Raja S.} and Julien Peltier and Thomas Macartney and Melanie Wightman and Alan Prescott and Robert Gourlay and Matthias Trost and Dario Alessi and Athanasios Karapetsas",
note = "This work was supported by the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.) and MC_UU_12016/5 (to MT)] and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.)",
year = "2019",
month = "10",
day = "30",
doi = "10.1042/BCJ20190608",
language = "English",
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journal = "Biochemical Journal",
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publisher = "Portland Press",
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TY - JOUR

T1 - Phosphoproteomics reveals that the hVPS34 regulated SGK3 kinase specifically phosphorylates endosomal proteins including Syntaxin-7, Syntaxin-12, RFIP4 and WDR44

AU - Malik, Nazma

AU - Nirujogi, Raja S.

AU - Peltier, Julien

AU - Macartney, Thomas

AU - Wightman, Melanie

AU - Prescott, Alan

AU - Gourlay, Robert

AU - Trost, Matthias

AU - Alessi, Dario

AU - Karapetsas, Athanasios

N1 - This work was supported by the Medical Research Council [grant number MC_UU_12016/2 (to D.R.A.) and MC_UU_12016/5 (to MT)] and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA, to D.R.A.)

PY - 2019/10/30

Y1 - 2019/10/30

N2 - 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 up-regulated 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 four 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 inefficiently 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 a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the 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.

AB - 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 up-regulated 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 four 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 inefficiently 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 a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the 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.

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KW - Phosphoproteomics

KW - phosphoinositide 3-kinase

KW - Akt

KW - syntaxins

KW - phosphoproteomics

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U2 - 10.1042/BCJ20190608

DO - 10.1042/BCJ20190608

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