Abstract
Activation of protein kinase C (PKC)-alpha decreases normal and neoplastic cell proliferation by inhibiting epidermal growth factor receptor (EGFR)-related signaling. The molecular interactions upstream to PKC-alpha that influence its suppression of EGFR, however, are poorly understood. We have found that caveolin-1, tetraspanin CD82, and ganglioside GM3 enable the association of EGFR with PKC-alpha, ultimately leading to inhibition of EGFR signaling. GM3- and CD82-induced inhibition of EGFR signaling requires PKC-alpha translocation and serine/threonine phosphorylation, which eventually triggers EGFR Thr654 phosphorylation and receptor internalization. Within this ordered complex of signaling molecules, the ability of CD82 to associate with PKC-alpha requires the presence of caveolin-1, whereas the interaction of caveolin-1 or PKC-alpha with EGFR requires the presence of CD82 and ganglioside GM3. Disruption of the membrane with methyl-beta-cyclodextrin dissociates the EGFR/GM3/caveolin-1/CD82/PKC-alpha complex and prevents the inhibitory effect of PKC-alpha on EGFR phosphorylation, suggesting that caveolin-1, CD82, and ganglioside interact with EGFR and PKC-alpha within intact cholesterol-enriched membrane microdomains. Given the role of these membrane molecules in suppressing EGFR signaling, up-regulation of GM3, caveolin-1, and CD82 function may be an effective adjunctive therapy for treating epithelial cell malignancies.
Highlights
Epidermal growth factor receptor (EGFR) signaling critically regulates cell proliferation and migration in normal and neoplastic cells
Previous studies by our laboratory and others have established that protein kinase C (PKC)-a phosphorylates and down-regulates EGFR [1], GM3 induces the association of caveolin-1 with EGFR and downregulates its signaling [20], and CD82 coimmunoprecipitates with EGFR to suppress its activation [9, 10, 38]
We show that all of these components associate in an ordered fashion: CD82 associates with EGFR and allows caveolin-1 to associate with EGFR; caveolin1 enables PKC-a to associate with CD82 and with EGFR; and all of these associations are potentiated by ganglioside GM3, which enables PKC-a translocation to the membrane and is required for PKC-a and CD82 to associate with EGFR
Summary
Epidermal growth factor receptor (EGFR) signaling critically regulates cell proliferation and migration in normal and neoplastic cells. The demonstration that EGFR number and activity are increased in many malignancies has promoted the development as cancer therapy of anti-EGFR antibodies and small molecules that target EGFR activity. The mechanisms that regulate EGFR signaling at the membrane level are incompletely understood. Much attention has focused on ligand-induced EGFR signaling and EGFR transactivation, but little attention has been paid to negative feedback on EGFR signaling by protein kinase C (PKC)-a activation [1]. Activated PKC-a translocates from the nucleus to the membrane, Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Departments of Dermatology and Pediatrics, Northwestern University Feinberg School of Medicine, 676 North St. Clair Avenue, Suite 1600, Chicago, IL 60611. Phone: 312-695-3721; Fax: 312-695-0664; E-mail: apaller@ northwestern.edu
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