Involvement of calpain-7 in epidermal growth factor receptor degradation via the endosomal sorting pathway
Involvement of calpain-7 in epidermal growth factor receptor degradation via the endosomal sorting pathway
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calpain-7 通过内体分选途径参与表皮生长因子受体降解
DOI：
10.1111/febs.12886
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Masatoshi Maki
中科院分区：
文献类型：
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作者：
Yuki Maemoto;Yasuko Ono;Satomi Kiso;Hideki Shibata;Terunao Takahara;Hiroyuki Sorimachi;Masatoshi Maki
钙蛋白酶-7（CAPN 7）是一种独特的细胞内半胱氨酸蛋白酶，具有微管相互作用和运输（MIT）结构域的串联重复序列，缺乏五EF手结构域。尽管CAPN 7的MIT结构域先前被证明与转运（ESCRT）-III和ESCRT-III相关蛋白所需的内体分选复合物的子集相互作用，包括带电多泡体蛋白1和增加的钠耐受性（IST）1，但蛋白酶参与膜运输的知识有限。在本研究中，与对照细胞相比，我们发现在CAPN 7敲除的HeLa细胞和从CAPN 7敲除（Capn 7 −/−）小鼠建立的小鼠胚胎成纤维细胞中，表皮生长因子受体（EGFR）降解轻微延迟。野生型CAPN 7而非CAPN 7的蛋白酶失活突变体（CAPN 7 C290 S）的再表达导致EGFR降解速率的恢复。我们通过免疫荧光显微镜分析发现，在HeLa细胞中，表皮生长因子刺激后，与CAPN 7的蛋白酶失活突变体[单体绿色荧光蛋白（mGFP）-CAPN 7 C290 S]融合的单体GFP被动员到EGFR阳性内体。虽然mGFP-CAPN 7 C290 S对EGFR降解表现出显性负效应，但mGFP-CAPN 7 C290 S中MIT结构域的缺失突变体不具有这种特性，这表明MIT结构域和ESCRT蛋白之间的相互作用对CAPN 7的功能很重要。此外，我们发现表皮生长因子刺激诱导IST 1从胞质溶胶易位到EGFR和mGFP-CAPN 7 C290 S中均呈阳性的内体。当IST 1被敲除时，mGFP-CAPN 7 C290 S失去了与EGFR的共定位。这些结果首次证明CAPN 7的蛋白水解活性对于利用ESCRT系统的一部分通过内体分选途径加速EGFR降解是重要的。（查看互动）EGFR，CAPN 7和IST 1通过荧光显微镜共定位（View interaction）EEA 1和CAPN 7通过荧光显微镜共定位（View interaction）CAPN 7和LAMP 1通过荧光显微镜共定位（View interaction）
Calpain‐7 (CAPN7) is a unique intracellular cysteine protease that has a tandem repeat of microtubule interacting and trafficking (MIT) domains and lacks a penta‐EF‐hand domain. Although the MIT domains of CAPN7 were previously shown to interact with a subset of endosomal sorting complex required for transport (ESCRT)‐III and ESCRT‐III‐related proteins, including charged multivesicular body protein 1 and increased sodium tolerance (IST)1, knowledge of the involvement of the protease in membrane trafficking has been limited. In the present study, compared with control cells, we found that epidermal growth factor receptor (EGFR) degradation was mildly delayed in CAPN7‐knockdown HeLa cells and mouse embryonic fibroblast cells established from CAPN7 knockout (Capn7−/−) mice. Re‐expression of wild‐type CAPN7 but not a protease‐inactive mutant of CAPN7 (CAPN7C290S) resulted in a recovery of the rate of EGFR degradation. We found, by immunofluorescence microscopic analysis, that monomeric GFP fused with the protease‐inactive mutant of CAPN7 [monomeric green fluorescent protein (mGFP)‐CAPN7C290S] was mobilized to EGFR‐positive endosomes upon epidermal growth factor stimulation in HeLa cells. Although mGFP‐CAPN7C290Sexhibited dominant‐negative effects on EGFR degradation, a deletion mutant of MIT domains in mGFP‐CAPN7C290Sdid not have such properties, suggesting that the interaction between the MIT domains and ESCRT proteins is important for the function of CAPN7. Moreover, we found that epidermal growth factor stimulation induces translocation of IST1 from the cytosol to endosomes positive in both EGFR and mGFP‐CAPN7C290S. When IST1 was knocked down, mGFP‐CAPN7C290Slost its co‐localization with EGFR. These results demonstrate for the first time that the proteolytic activity of CAPN7 is important for the acceleration of EGFR degradation via the endosomal sorting pathway utilizing a part of the ESCRT system.Structured digital abstractEGFR and CAPN7 colocalize by fluorescence microscopy (View interaction)EGFR, CAPN7 and IST1 colocalize by fluorescence microscopy (View interaction)EEA1 and CAPN7 colocalize by fluorescence microscopy (View interaction)CAPN7 and LAMP1 colocalize by fluorescence microscopy (View interaction)