Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development. Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis. The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas. The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs.

中文翻译：

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磷酸酶 Shp1 与 cortactin 相互作用并使其去磷酸化以抑制侵袭伪足功能

侵袭伪足是与伴随癌症侵袭的细胞外基质降解相关的基于肌动蛋白的细胞膜突起。阐明导致侵袭伪足形成和活动的分子机制对于预防肿瘤扩散和生长至关重要。已知蛋白酪氨酸激酶如 Src 可调节侵袭伪足组装，但对蛋白酪氨酸磷酸酶在此过程中的作用知之甚少。在这些酶中，我们选择了酪氨酸磷酸酶 Shp1 来研究它在侵袭伪足组装中的潜在作用，因为它参与了癌症的发展。共免疫沉淀和免疫荧光研究用于鉴定控制入侵伪足活性的 Shp1AQ 的新底物。本研究中鉴定的 Shp1 底物 cortactin 的磷酸化水平，通过免疫沉淀、体外磷酸酶和蛋白质印迹分析评估。短干扰 RNA 和在 A375MM 黑色素瘤细胞中表达的 Shp1 催化死亡突变体被用来评估特异性 Shp1 介导的 cortactin 去磷酸化的作用。通过细胞外基质降解测定和体内转移小鼠模型研究了直接结合和调节 Shp1 的甘油磷酸肌醇的抗侵袭特性。数据显示，Shp1 被招募到入侵伪足并促进皮质素在 421 位酪氨酸处的去磷酸化，导致黑色素瘤癌细胞降解细胞外基质的能力减弱。对照包括使用短干扰 RNA 和催化死亡突变体，防止 cortactin 去磷酸化，从而减少黑色素瘤细胞的细胞外基质降解。此外，磷酸肌醇代谢物甘油磷酸肌醇促进了 Shp1 在侵袭伪足的定位，从而促进了 cortactin 去磷酸化。这在体外和黑色素瘤的体内模型中都损害了侵袭伪足功能和肿瘤传播。这里报道的主要发现是，cortactin 是酪氨酸磷酸酶 Shp1 的特异性底物，其磷酸化/去磷酸化会影响侵袭伪足的形成，因此会影响黑色素瘤细胞侵入细胞外基质的能力。