Membrane Proximal ERK Signaling Is Required for M-calpain Activation Downstream of Epidermal Growth Factor Receptor Signaling

Abstract

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 deadhesion. 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 adhesionrelated substrates.

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

“…However, ERKs extranuclear component is just as important. It has been estimated that half of the ERKs content remains in the cytoplasm after stimulation [62] and processes such as the formation of cell-matrix contacts, [63] adhesion, [64] endosomal traffic, [65] Golgi fragmentation, [66] and anti-apoptotic signaling [67] are dependent on ERK extranuclear activity. It fact, nearly half of the $180 proteins thus far identified as ERKs substrates, are non-nuclear proteins.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Calpain cleavage of talin here is a rate limiting step in disassembly of other focal adhesion components including zyxin, paxillin and vinculin [190]. Recent studies have demonstrated that growth factor stimulation of calpain activity and cellular deadhesion is dependent upon ERK [191,192]. Calpain is directly phosphorylated by ERK in response to EGF stimulation and this increases calpain's proteolytic activity [96].…”

Regulating cell migration: calpains make the cut

“…Together, these data suggest that calcium and growth-factormediated phosphorylation can independently activate calpains in an isoform-specific fashion. Interestingly, only membraneproximal calpain 2 is activated by ERK-mediated phosphorylation (Glading et al, 2001), which suggests that there are alternative modes of activation for certain calpain 2 subpopulations.…”