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

Angela Glading,Florian Überall,Stephen M Keyse,Douglas A Lauffenburger,Alan Wells

doi:10.1074/jbc.m008847200

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 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.

Highlights

The individual biophysical processes of extension, adhesion, de-adhesion, and contraction must be finely regulated in a temporal and spatial manner to enable productive fibroblast motility [3]
We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated extracellular signal-regulated kinase (ERK) activation is necessary for epidermal growth factor (EGF)induced motility
Calpain Is Activated by an Internalization-deficient EGFR, cЈ973—NR6 fibroblasts expressing both wild type (WT) and internalization-deficient EGFR were used in the Boc-LM-CMAC calpain activity assay. cЈ973 NR6 cells exhibited calpain activity equivalent to that seen in WT cells (Fig. 2)

Summary

Introduction

The individual biophysical processes of extension, adhesion, de-adhesion, and contraction must be finely regulated in a temporal and spatial manner to enable productive fibroblast motility [3]. 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. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity.

Results

Conclusion