Douglas A. Lauffenburger

Epidermal Growth Factor Receptor Activation of Calpain Is Required for Fibroblast Motility and Occurs via an ERK/MAP Kinase Signaling Pathway

To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cγ signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation.

Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 ras in Fibroblasts

Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc·Grb2·Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-α, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.

Effect of Epidermal Growth Factor Receptor Internalization on Regulation of the Phospholipase C-γ1 Signaling Pathway

The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-α (TGFα) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGFα-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-γ1 (PLC-γ1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGFα stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP2) hydrolysis over those stimulated by TGFα. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP2 hydrolysis, PLC-γ1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-γ1 phosphorylation and PIP2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-γ1 to its substrate in endocytic trafficking organelles.

Growth factor-induced cell motility in tumor invasion.

Tumor progression to the invasive and metastatic states dramatically enhances the morbidity and mortality of cancer. Rational therapeutic interventions will only be possible when we understand the molecular mechanisms governing the cell behavior underlying this transformation. For invasion, a subpopulation of tumor cells must recognize the extracellular matrix barrier, modify the barrier, migrate through the barrier, and then proliferate in the adjacent but ectopic locale. Prevention of any one of these steps would prevent invasion, but determining the most sensitively dysregulated step should provide the most promising therapeutic index. In many invasive tumors, upregulation of active motility is stimulated by growth factor receptor signaling, the EGF receptor being the most frequently implicated. Two key downstream molecular switches, PLC n and m-calpain, are required for growth factor-induced motility but not basal, matrix-stimulated motility. Inhibition of either of these enzymes blocks in vitro and in vivo invasion of prostate, breast, and bladder carcinomas and glioblastomas. These represent novel and potentially selective targets for drug development. Future advances in the imaging of tumors in animals and ex vivo organ culture systems should provide additional new targets.

Epidermal growth factor receptor signaling in tissues

A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptors cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.