人肥大细胞中的 SCF/KIT 轴：Capicua 作为有效的 KIT 抑制剂，ERK 占主导地位而 PI3K 被抑制。

The SCF/KIT axis in human mast cells: Capicua acts as potent KIT repressor and ERK predominates PI3K.

机构信息

Institute of Allergology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.

Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Immunology and Allergology IA, Berlin, Germany.

出版信息

Allergy. 2022 Nov;77(11):3337-3349. doi: 10.1111/all.15396. Epub 2022 Jun 14.

DOI:10.1111/all.15396

PMID:35652819

Abstract

BACKGROUND

The SCF/KIT axis regulates nearly all aspects of mast cell (MC) biology. A comprehensive view of SCF-triggered phosphorylation dynamics is lacking. The relationship between signaling modules and SCF-supported functions likewise remains ill-defined.

METHODS

Mast cells were isolated from human skin; upon stimulation by SCF, global phosphoproteomic changes were analyzed by LC-MS/MS and selectively validated by immunoblotting. MC survival was inspected by YoPro; BrdU incorporation served to monitor proliferation. Gene expression was quantified by RT-qPCR and cytokines by ELISA. Pharmacological inhibitors were supplemented by ERK1 and/or ERK2 knockdown. CIC translocation and degradation were studied in nuclear and cytoplasmic fractions. CIC's impact on KIT signaling and function was assessed following RNA interference.

RESULTS

≈5400 out of ≈10,500 phosphosites experienced regulation by SCF. The MEK/ERK cascade was strongly induced surpassing STAT5 > PI3K/Akt > p38 > JNK. Comparison between MEK/ERK's and PI3K's support of basic programs (apoptosis, proliferation) revealed equipotency between modules. In functional outputs (gene expression, cytokines), ERK was the most influential kinase. OSM and LIF production was identified in skin MCs. Strikingly, SCF triggered massive phosphorylation of a protein not associated with KIT previously: CIC. Phosphorylation was followed by CIC's cytoplasmic appearance and degradation, the latter sensitive to protease but not preoteasome inhibition. Both shuttling and degradation were ERK-dependent. Conversely, CIC-siRNA facilitated KIT signaling, functional outputs, and survival.

CONCLUSION

The SCF/KIT axis shows notable strength in MCs, and MEK/ERK as most prominent module. An inhibitory circuit exists between KIT and CIC. CIC stabilization in MCs may turn out as a therapeutic option to interfere with allergic and MC-driven diseases.

摘要

背景

SCF/KIT 轴调节 mast cell（MC）生物学的几乎所有方面。缺乏对 SCF 触发的磷酸化动态的全面了解。信号模块与 SCF 支持的功能之间的关系同样也没有明确界定。

方法

从人皮肤中分离出 mast cells；通过 SCF 刺激，通过 LC-MS/MS 分析全局磷酸蛋白质组变化，并通过免疫印迹选择性验证。通过 YoPro 检查 MC 的存活；通过 BrdU 掺入监测增殖。通过 RT-qPCR 定量基因表达，通过 ELISA 定量细胞因子。用 ERK1 和/或 ERK2 敲低补充药理学抑制剂。研究 CIC 易位和降解在核和细胞质部分。评估 RNA 干扰后 CIC 对 KIT 信号和功能的影响。

结果

约 5400 个磷酸化位点中有约 10500 个受到 SCF 的调节。MEK/ERK 级联反应强烈诱导，超过 STAT5 > PI3K/Akt > p38 > JNK。比较 MEK/ERK 和 PI3K 对基本程序（凋亡、增殖）的支持作用，发现模块之间具有等效性。在功能输出（基因表达、细胞因子）中，ERK 是最具影响力的激酶。在皮肤 MC 中鉴定出 OSM 和 LIF 的产生。令人惊讶的是，SCF 引发了先前与 KIT 无关的一种蛋白质的大量磷酸化：CIC。磷酸化后，CIC 出现在细胞质中并降解，后者对蛋白酶敏感，但对蛋白酶体抑制不敏感。穿梭和降解都依赖于 ERK。相反，CIC-siRNA 促进了 KIT 信号、功能输出和存活。