Glading A, Uberall F, Keyse S M, Lauffenburger D A, Wells A
Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
J Biol Chem. 2001 Jun 29;276(26):23341-8. doi: 10.1074/jbc.M008847200. Epub 2001 Apr 23.
Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as de-adhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesion-related substrates.
信号定位在引导细胞命运方面至关重要，尤其是在多效性信号通路中。促进细胞迁移、增殖和分化的细胞外信号调节激酶（ERK）/微管相关蛋白激酶存在于细胞核和整个细胞质中。最近研究表明，ERK的核转位是转录变化和细胞增殖所必需的。然而，细胞质信号传导的细胞后果尚未明确。我们探究了细胞质中，特别是膜近端的ERK信号是否参与生长因子诱导的细胞运动。我们之前已经证明，表皮生长因子受体（EGFR）介导的ERK激活下游的M-钙蛋白酶活性增加是表皮生长因子（EGF）诱导运动所必需的。在多种细胞类型的细胞核、细胞质和质膜相关区室中也发现了钙蛋白酶同工型。我们现在采用细胞工程方法来控制上游EGFR和ERK活性的定位，以研究上游信号位点对下游钙蛋白酶活性的空间效应。通过差异配体诱导的内化和运输受限的受体变体，我们发现钙蛋白酶活性仅由质膜受限的活化EGFR触发，而不是由内化的（尽管仍然活跃）EGFR触发。用膜靶向的ERK1和ERK2转染细胞，它们隔离内源性ERK，这些细胞表现出正常的EGF诱导的钙蛋白酶活性。转染一种无活性的ERK磷酸酶（MKP-3/Pyst1），它将ERK隔离在细胞质中，可阻止钙蛋白酶激活以及细胞去粘附。这些数据强烈表明，EGF诱导的钙蛋白酶活性可以在膜近端EGFR介导的ERK信号位点附近增强，这为钙蛋白酶活性如何被调节和靶向以增强其对粘附相关底物的作用提供了见解。
