Interaction of Paxillin with Poly(A)-Binding Protein 1 and Its Role in Focal Adhesion Turnover and Cell Migration

Abstract

We have previously identified poly(A)-binding protein 1 (PABP1) as a ligand for paxillin and shown that the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling. By targeting the paxillin-binding subdomain sequences in PABP1, we have generated mutants of PABP1 that do not bind to cellular paxillin. Here we report that paxillin association is necessary for efficient nuclear export of PABP1 and that RNA interference of paxillin drives the nuclear accumulation of PABP1. Furthermore, ablation of paxillin-PABP1 association impeded a number of indices of cell motility including spreading on fibronectin, cell migration on twodimensional matrices, and transmigration in Boyden chambers. These data indicate that PABP1 must associate with paxillin in order to be efficiently transported from the nucleus to the cytoplasm and that this event is necessary for cells to remodel their focal adhesions during cell migration.The leading edge or lamellipodium of migrating cells is the point at which integrins engage the extracellular matrix, an interaction that leads to integrin clustering into an array of small multiprotein focal complexes (31) containing an array of structural and signaling proteins that act to establish and maintain the polarity of the migrating cell. The 68-kDa protein paxillin is an abundant component of focal complexes at the leading edge of migrating cells. In addition to binding directly to integrin cytodomains, paxillin contains numerous proteinbinding modules that interact with a variety of structural and signaling proteins and is therefore classified as a molecular adaptor or scaffold protein (23). Paxillin has an N-terminal region with five leucine-rich motifs, termed LD domains, and a C-terminal portion with four tandem LIM domains. The LIM domains contain information for targeting paxillin to focal complexes and can bind directly to tubulin (9). LD domains are protein-protein interaction motifs with the consensus sequence LDXXLLXXL, and these mediate the interaction of paxillin with a number of proteins that regulate cell migration. LD domains seem to display some degree of selectivity with respect to the ligands they recruit; LD1 binds integrin-linked kinase and the actin-binding proteins vinculin and actopaxin, while LD2 associates with focal adhesion kinase (FAK) and the ARF-GAP protein, p95PKL (23). Based on mutational analysis, Turner and coworkers (23) have identified the regions of proteins that associate with the various LD domains and termed them paxillin-binding subdomains or PBSs.Using a proteomic approach, we have recently identified an association between paxillin and the mRNA-binding protein, PABP1. Moreover, the paxillin-PABP1 complex undergoes nucleocytoplasmic shuttling and is localized to sites of translation in the perinuclear endoplasmic reticulum and at the leading edge of migrating cells (29). PABP1 consists of an N-terminal portion that contains four tandem RNA-binding motifs (RRM) and a C-terminal region with homology to an ubiquitin E3 ligase, called HYD (Fig. 1A) (12). ...

Evidence that Poly(A) Binding Protein C1 Binds Nuclear Pre-mRNA Poly(A) Tails

“…In humans, at least four separate PABPC genes and four pseudogenes have been identified (40). PABPC1 influences mRNA translation and decay (18,20,23,27,29,30,50,(54)(55)(56), and it shuttles between the nucleus and cytoplasm of at least some mammalian cells (1,57,58). Consistent with the preferential compartmentalization of PABPN1 to the nucleus and PABPC1 to the cytoplasm, a physical interaction has been detected between PAP and PABPN1 but not PABPC1 (28).…”

“…Although PABPC1 has been characterized as exclusively cytoplasmic in selected HeLa and Cos cell lines (48), more recent studies demonstrate that PABPC1 shuttles between the nucleus and cytoplasm in at least some HeLa and NIH 3T3 cell lines (1,57,58). We utilized cell fractionation and rabbit polyclonal anti-PABPC1 (33) to determine if PABPC1 was present in the nuclear fraction of HeLa CCL2 cells, since we used these cells in subsequent studies (see below).…”

“…In either scenario, PABPC1 may also be involved in the localization of particular mRNAs to specific regions of the cytoplasm. For example, PABPC1 was recently shown to bind directly to paxillin, which is an abundant protein of focal complexes at the leading edges of migrating cells (57,58). The PABPC1-paxillin complex localizes to the perinuclear endoplasmic reticulum and the leading edge of the migrating cell plasma membrane in mouse fibroblasts.…”

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

“…Paxillin is involved in many processes including cell adhesion and spreading. 37,[48][49][50][51] Studies on paxillin functions in paxillin null or knockdown cells using different ECM substrates had conflicting results. 28,49,50,52 Therefore, we hypothesized that LPXN and paxillin might be differentially involved in integrin signaling induced by different ECM proteins.…”

Phosphoproteomic analysis of Her2/neu signaling and inhibition

“…AIP1͞Alix inhibited EGFR internalization and degradation; therefore, we hypothesize that Pdcd-6͞Alg-2 protects Her2 from Cbl-mediated degradation. Similarly, poly(A)-binding protein cytoplasmic 1 (PABPC1) binds and colocalizes with paxillin, and disruption of this interaction inhibited cell migration (28). In 3T3-Her2 cells, Pabpc1 showed increased phosphorylation (Table 1), and paxillin showed borderline decreased phosphorylation (fold change of 0.68; Table 2), suggesting that Her2-induced phosphorylation of Pabpc1 may affect focal adhesions.…”