Editors' ChoiceDevelopment

Pseudosubstrate-Mediated Inhibition Promotes Migration

Sci. Signal.  19 Nov 2013:
Vol. 6, Issue 302, pp. ec283
DOI: 10.1126/scisignal.2004915

Epithelial cell adhesion molecule (EpCAM) is present on the surface of epithelia and various tumors and engages in homophilic interactions that mediate cell-cell adhesion. EpCAM contains two extracellular epidermal growth factor domains, a transmembrane domain, and a short cytoplasmic tail and has been associated with increased proliferation and migration in cultured cancer cell lines. The proliferative effect of EpCAM has been attributed to the proteolytic release of the cytoplasmic domain, which translocates to the nucleus and contributes to a transcriptional activator complex. Maghzal et al. investigated the mechanism by which EpCAM promotes migration and found that a portion of the cytoplasmic tail of EpCAM enhanced cell migration by binding to and inhibiting novel protein kinase Cs (nPKCs), which are a subset of PKCs that are activated by diacylglycerol but, in contrast to classical PKCs, not Ca2+. Knockdown of EpCAM in Xenopus laevis (frog) embryos caused early lethality characterized by loss of C-cadherin in the epidermis followed by epidermal disintegration. Overexpression of C-cadherin rescued the loss of epithelial integrity in ectoderm explants from EpCAM-depleted embryos. Experiments with inhibition and overexpression of various regulators of cytoskeletal contractility suggested that the loss of EpCAM increased nPKC activity, thereby activating signaling through a MAPK (mitogen-activated protein kinase) pathway that resulted in the activation of myosin light chain kinase, increased myosin contractility, and endocytosis of C-cadherin. The 13 amino acids of the cytoplasmic tail closest to the membrane resembled the autoinhibitory pseudosubstrate motifs in the nPKCs PKCδ and PKCη, suggesting that this juxtamembrane motif (JM) might bind to and inhibit one of these nPKCs. Endogenous EpCAM and PKCδ coimmunoprecipitated from extracts of cultured colon cancer cells, and a tagged version of the EpCAM cytoplasmic tail pulled down PKCδ from Xenopus extracts. In vitro, the tagged EpCAM cytoplasmic tail and peptides corresponding to the human or zebrafish EpCAM JM bound to recombinant PKCδ and PKCη but not to a classical PKC (PKCβ). The EpCAM cytoplasmic tail inhibited PKCδ activity in vitro. Key conserved residues in this motif were required for strong binding to PKCδ and for recombinant EpCAM to rescue epithelial integrity and C-cadherin abundance in EpCAM-depleted embryos. The authors identified similar putative JM motifs in several human cell adhesion proteins and found that those from EphA4, ICAM1, and NrCAM bound to PKCδ and PKCη in vitro, suggesting that this mechanism of nPKC inhibition might be shared by other transmembrane proteins.

N. Maghzal, H. A. Kayali, N. Rohani, A. V. Kajava, F. Fagotto, EpCAM controls actomyosin contractility and cell adhesion by direct inhibition of PKC. Dev. Cell 23, 263–277 (2013). [PubMed]