Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation.
Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation.
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DOI：
10.1016/j.jbc.2022.101750
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Lichtenstein A
中科院分区：
文献类型：
--
作者：
Vega M;Chen Y;Shi Y;Gera J;Lichtenstein A
DEPTOR是一种在多发性骨髓瘤（MM）细胞中上调的48 kDa蛋白。DEPTOR抑制mTOR，并通过抑制负反馈环，促进AKT激活。我们先前鉴定了一种与MM细胞中的DEPTOR结合并诱导其蛋白酶体降解的化合物。为了确定降解的机制，在这里，我们筛选了药物诱导的翻译后修饰，并确定了丝氨酸235（S235）上DEPTOR的磷酸化减少。我们发现，S235磷酸模拟DEPTOR突变体是耐降解，证实了这种翻译后修饰的重要性。此外，在S235处具有丝氨酸至丙氨酸取代的DEPTOR突变体只能在同时蛋白酶体抑制后表达。因此，S235磷酸化调节DEPTOR稳定性。筛选DEPTOR相互作用组鉴定出USP-7去泛素酶与DEPTOR的缔合依赖于S235磷酸化。USP-7活性的抑制导致DEPTOR多聚遍在蛋白化和降解。扫描搜索表明，ERK 1可能是负责S235磷酸化，这是通过使用抑制剂，ERK 1敲低，并在体外激酶测定证实。ERK 1的抑制也下调AKT磷酸化。为了测试DEPTOR磷酸化是否介导这种串扰，MM细胞用WT或磷酸化模拟DEPTOR转染并暴露于ERK抑制剂。虽然WT DEPTOR对AKT磷酸化的抑制没有影响，但磷酸模拟物DEPTOR阻止了抑制。这些结果表明ERK 1通过DEPTOR的磷酸化维持MM细胞中的AKT活性。我们提出，DEPTOR依赖的串扰提供了MM细胞的活力促进信号（通过AKT）时，增殖刺激（通过ERK）。
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).
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