Membrane proximal ERK signaling is required for M-calpain activation downstream of epidermal growth factor receptor signaling

Localization of signaling is critical in directing cellular outcomes, espec ially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/ microtubule-associated protein kinase, which promotes cell m igration, proliferation, and differentiation is found in the nucleus and th roughout the cytoplasm. Recently, it has been shown that nuclear translocat ion 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, E RK signaling is involved in growth factor-induced cell motility, We previou sly have demonstrated that increased M-calpain activity downstream of epide rmal 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 compartmen ts in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine th e 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 plas ma 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 E RK in the cytoplasm prevented calpain activation as well as deadhesion. The se data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insi ghts about how calpain activity might be regu lated and targeted to enhance its effects on adhesion-related substrates.