Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation

J Biol Chem. 2022 Apr;298(4):101750. doi: 10.1016/j.jbc.2022.101750. Epub 2022 Feb 23.

Abstract

DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an in vitro kinase assay. Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).

Keywords: AKT; DEPTOR; ERK; mTOR; multiple myeloma.

MeSH terms

  • Humans
  • Intracellular Signaling Peptides and Proteins* / genetics
  • Intracellular Signaling Peptides and Proteins* / metabolism
  • MTOR Inhibitors / pharmacology
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Multiple Myeloma* / drug therapy
  • Multiple Myeloma* / metabolism
  • Mutation
  • Phosphorylation / drug effects
  • Proto-Oncogene Proteins c-akt* / genetics
  • Proto-Oncogene Proteins c-akt* / metabolism
  • Serine / metabolism
  • Signal Transduction

Substances

  • Intracellular Signaling Peptides and Proteins
  • MTOR Inhibitors
  • Serine
  • DEPTOR protein, human
  • MTOR protein, human
  • Proto-Oncogene Proteins c-akt
  • MAPK3 protein, human
  • Mitogen-Activated Protein Kinase 3