Regulation of paxillin family members during epithelial-mesenchymal transformation: a putative role for paxillin δ

Abstract

Epithelial-mesenchymal transformation (EMT) and the resulting induction of cell motility are essential components of tissue remodeling during embryonic development and wound repair, as well as tumor progression to an invasive metastatic phenotype. Paxillin, a multi-domain adaptor and phosphoprotein has previously been implicated in integrin signaling and cell motility. In this report we characterize a novel paxillin gene product, paxillin δ, generated from an evolutionarily conserved internal translation initiation site within the full-length paxillin mRNA. Paxillin δ, which lacks the key phosphorylation sites Y31 and Y118 as well as the ILK and actopaxin binding LD1 motif, exhibits a restricted distribution to epithelial cell types and is downregulated during TGF-β1-induced EMT of normal murine mammary gland (NMuMG) epithelial cells. Interestingly, Hic-5, a paxillin superfamily member, exhibits a reciprocal protein expression profile to paxillin δ. In addition, paxillin δ expression is maintained following NMuMG differentiation in a 3D collagen I gel while other focal adhesion components are downregulated. Paxillin δ protein expression coincided with reduced paxillin tyrosine phosphorylation in NMuMG cells and paxillin δ overexpression in CHO.K1 cells inhibited adhesion-mediated tyrosine phosphorylation of paxillin. Forced expression of paxillin δ in NMuMG cells suppressed cell migration whereas Hic-5 overexpression stimulated motility. Together our data support a role for paxillin δ as a naturally occurring functional antagonist of paxillin signaling potentially through suppression of a Crk-mediated pathway during processes associated with cell migration.

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…For example, when compared to α-paxillin, β-paxillin exhibits a reduced binding affinity for vinculin, but an equivalent affinity for FAK; the inverse has been shown to be true for γ-paxillin [87]. Additionally, β and γ-paxillin exhibit a more restricted expression profile in comparison to α-paxillin, being expressed primarily in cancer and monocytic cells, respectively [87,88]. The expression of the fourth paxillin isoform however, δ-paxillin (46 kDa), is enriched in epithelial cells while absent in mesenchymal cells [66,[86][87][88].…”

“…Additionally, β and γ-paxillin exhibit a more restricted expression profile in comparison to α-paxillin, being expressed primarily in cancer and monocytic cells, respectively [87,88]. The expression of the fourth paxillin isoform however, δ-paxillin (46 kDa), is enriched in epithelial cells while absent in mesenchymal cells [66,[86][87][88]. The δ-paxillin isoform is unique in comparison to the α and β transcriptional variants in that it is the result of an alternative translation initiation site located 132 amino acids downstream of the α-paxillin start codon [88].…”

“…The expression of the fourth paxillin isoform however, δ-paxillin (46 kDa), is enriched in epithelial cells while absent in mesenchymal cells [66,[86][87][88]. The δ-paxillin isoform is unique in comparison to the α and β transcriptional variants in that it is the result of an alternative translation initiation site located 132 amino acids downstream of the α-paxillin start codon [88]. This internally translated paxillin isoform differs structurally in its N-terminal domain, lacking the proline-rich and SH2-binding site containing LD1 motif found in α-paxillin [88].…”

“…The δ-paxillin isoform is unique in comparison to the α and β transcriptional variants in that it is the result of an alternative translation initiation site located 132 amino acids downstream of the α-paxillin start codon [88]. This internally translated paxillin isoform differs structurally in its N-terminal domain, lacking the proline-rich and SH2-binding site containing LD1 motif found in α-paxillin [88]. Functionally speaking, there is evidence to suggest that this truncated isoform of paxillin acts antagonistically to the function of the wildtype, full-length α-paxillin (hereafter referred to as paxillin), to limit the inappropriate trans-differentiation and cell migration that is required for epithelial cell invasion [66,86,87].…”

“…Whereas the expression profile of leupaxin is exceedingly limited, the expression of Hic-5 (Hydrogen Peroxide-Inducible Clone-5), also known as ARA55 (Androgen receptor Activator 55) has been shown in platelets, cells of mesenchymal origin as well as stromal and smooth muscle tissue layers; Hic-5 expression is altogether absent in epithelial cells [66,87,94]. Hic-5, a 55 kDa protein that is ≈ 57% homologous to paxillin, was originally identified as a TGF-β and hydrogen peroxide-inducible gene in the mouse osteoblastic cell line MC3T3 [66,88,94]. Although has been shown to have a similar function to paxillin, there is a growing amount of evidence that suggests a role for Hic-5 as a natural antagonist of paxillin by virtue of its ability to ablate paxillin signaling via competition of shared LD binding partners PTP-PEST and FAK, and suppression of downstream paxillin tyrosine phosphorylation [88].…”