Paxillin: a crossroad in pathological cell migration

The PXN gene is highly conserved among species: orthologs of human PXN [8] have been identified in 168 organisms [9]. Human PXN gene is located on chromosome 12q24 and contains, at least, 20 exons within its 55 kbp length (Fig. 2a). Four isoforms derived from alternative splicing have been described: Isoform 1(α) represents canonical paxillin [9]. Isoform 2(β) results from the insertion of exon 15, and isoform 3(γ) derives from the use of an alternative 5′-donor site for exon 16. Isoform 4(δ) shares isoform 1 structure, except for a shorter N-terminal domain, derived from the use of an alternative transcription initiation site in exon 2 [10] (Fig. 2). Paxillin isoforms exhibit different expression patterns, α form being the most widely expressed, whereas the expression of the β isoform is more restricted, and that of paxillin γ is related to specific cell differentiation stages [11, 12]. Paxillin δ seems to be exclusively expressed in epithelial cells [10]. Remarkably, at least 26 additional alternative splice variant messenger RNAs (mRNAs) derived from automated computational analysis are predicted in the Genebank (Additional file 1), suggesting that paxillin might have additional isoforms whose functional role remains to be established.

Bioinformatic analysis of PXN promoter region (Ensembl Regulatory Element ENSR00000091396) results in a 3401-bp region, 1271 bp upstream and 2130 bp downstream the transcription start site (TSS), and contains a number of putative binding sites for several transcription factors including AP-1, CREB, STAT3, c-Jun, and c-Myc [13]. The regulatory elements which have been experimentally validated are shown in Fig. 2b [14‐16].

MicroRNAs (miRNAs) are small noncoding RNAs that serve as negative regulators of gene expression [17] and have been suggested as key players in the regulation of tumor cell proliferation and invasion [18]. Among these, miR-212, miR-132, miR-145, miR-137, and miR-218 have been shown to target the 3′UTR region of PXN. Furthermore, their levels are inversely correlated with PXN expression in gastric cancer cell lines, colorectal cancer, as well as in non-small cell lung cancer [19‐23]. Therefore, the expression of these miRNAs has been proposed as predictive of tumor malignancy and as therapeutic factors.

Paxillin is an adapter protein containing five repetitive leucine-rich LD (Leu-Asp) motifs located at the N-terminus (LD1 to LD5) and four cystein-histidine-enriched LIM domains at the C-terminus. Additionally, it contains a proline-rich sequence that anchors SH3-containing proteins, located within the N-terminus, as well as numerous serine and tyrosine residues throughout the protein, which bind to SH2 domains (Fig. 3).

The LD motifs form amphipathic helices with leucine residues laterally aligned on the helix backbone, thus generating a hydrophobic patch surrounded by negatively charged aspartic and glutamic acid residues [24]. LD domains provide docking sites for FA-associated proteins including tyrosine kinases of the Src family, focal adhesion kinase (FAK), actopaxin, vinculin, talin, integrin-linked kinase (ILK), p21-activated kinase (PAK), as well as the ARF-interacting adapters, G-protein-coupled receptor kinase-interacting proteins (GIT1/GIT2), ADP-ribosylation-interacting protein (ARF), and GTPase-activating proteins (GAPs). FAK interacts with paxillin at an evolutionary preserved LD consensus motif (LDLLXXLL), identified as the binding site for FAK and vinculin [25]. However, in the absence of the four C-terminal LIM domains, the binding of FAK and vinculin to the LD domains is insufficient for directing paxillin to the proper location. In fact, the C-terminal domain of FAK contains a ~150 amino acid “focal adhesion targeting (FAT) region” as well as binding sequences for paxillin and talin. Moreover, although FAK is recruited from the cytoplasm to adhesion sites by paxillin, FAK interaction with N-terminal paxillin domains is not required for paxillin localization at focal adhesions [26]. Particularly, the LD4 domain has been shown to anchor signaling proteins such as GIT1 and paxillin kinase linker p95 in addition to FAK. The proline-rich sequence at the N-terminal also provides a binding site for SH3 domains of the Src family and associates with the tyrosine phosphatase PTP-PEST known to regulate cell spreading and migration [5, 27]. Interestingly, the LD motifs also serve as docking sites for the E6 oncoprotein of bovine papillomavirus type 1, whose binding to paxillin is closely associated with disruption of the actin cytoskeleton and cell transformation by papillomavirus E6 oncoproteins [28]. Noticeably, differences between paxillin isoforms reside at the N-terminal region: Isoforms 2(β) and 3(γ) contain, respectively, 34 and 48 amino acid inserts downstream the LD4 motif [11]. Isoform 4(δ) has a shorter N-terminus than isoform 1(α), lacking the LD1 motif, the proline-rich region, and the key phosphorylation sites Tyr31 and Tyr118 [10].

The C-terminal region of paxillin is common to all isoforms and contains four cystein-histidine-enriched LIM domains, which are involved in protein-protein interactions, and function as an anchor to the plasma membrane. Specifically, the LIM3 domain is responsible for targeting paxillin to FA sites. Of notice, the serine/threonine phosphorylation at LIM2/3 domains cooperatively potentiates the localization of paxillin to FAs (Fig. 3) [26]. Paxillin also associates with the adapter protein 47 (GAG-CRK) [29], a convergence molecule for signals resulting from cell adhesion as well as from the activation of growth factor receptors, which interacts with proteins involved in the regulation of the cytoskeleton and tumor metastasis [27, 30].

Integrin binding to ECM promotes paxillin phosphorylation and recruitment of proteins involved in the assembly of FAs, including talin, vinculin, tensin, and FAK. Tyr phosphorylation of paxillin provides a scaffold for the recruitment of tyrosine kinases FAK and Src, which are activated as a result of adhesion or growth factor stimulation. The subsequent phosphorylation at Tyr88 and Tyr118 by Src [1, 32], as well as at Tyr118 and Tyr31 at the N-terminus by FAK, allows paxillin interaction with downstream effectors, such as p130cas, and the transduction of external signals into cellular responses mediated by mitogen-activated protein kinases (MAPKs) (Fig. 4). In addition to Src, paxillin Tyr118 binding to CRKL SH-2 domains and thereby to a Rac1 signaling complex activated by DOCK180 has also been described [33]. Although in vitro studies have shown Tyr118 to be the main residue for paxillin phosphorylation by FAK, such phosphorylation is not essential for paxillin localization to focal adhesions. In fact, the binding site for FAK is located at the N-terminal domain, whereas the sequence for localization at focal adhesions resides in the C-terminal region of paxillin. The interaction with FAK however increases the efficiency of paxillin phosphorylation, in addition to directing the phosphorylation of additional tyrosine residues [34]. Since diverse stimuli such as lysophosphatidic acid or platelet-derived growth factor promote Tyr phosphorylation of paxillin and FAK in distinct cell types during spreading, the phosphorylation of paxillin by FAK has been suggested to mediate transduction from cell adhesion to the reorganization of the cytoskeleton required for cell movement [35]. Tyrosine phosphorylation of paxillin has been shown to regulate both the assembly and the turnover of adhesion complexes; however, the significance of this process for adhesion dynamics is still unclear. Whereas phosphorylated paxillin seems to enhance lamellipodial protrusions, non-phosphorylated paxillin has been found essential for fibrillar adhesion formation and fibronectin fibrillogenesis. Recent data show that FAK preferentially interacts with phospho-tyrosine paxillin, which is implicated in high turnover of focal complexes and translocation of FAs [32].

The N-terminal domain of paxillin contains a number of serine/threonine phosphorylation sites, which become phosphorylated during cell migration. Recent studies suggest that paxillin turnover, including its degradation by the proteasome, is also regulated by serine phosphorylation during migration. Among the kinases that phosphorylate paxillin at Ser/Thr residues, the phosphorylation of Ser178 by JUN N-terminal kinase (JNK) is required for cell migration [36, 37]. On this line, Ser126 phosphorylation by the sustained activation of Raf/MEK/ERK, and of Ser130 by glycogen synthase kinase 3 (GSK3), has been shown to induce the translocation of paxillin from FAs to the cytosol (Fig. 4) [38]. Furthermore, paxillin phosphorylation at Ser126 and Ser130 has also been found to regulate cytoskeletal rearrangement and cell movement in lipopolysaccharide-stimulated macrophages, through an extracellular regulated kinase (ERK)/GSK3 dual-kinase mechanism, which requires ERK-mediated phosphorylation of Ser130 for the subsequent phosphorylation of Ser126 by GSK3 [33]. Additionally, p21-activated kinase (PAK)-induced phosphorylation of paxillin at Ser273 has been identified as a critical regulator of Rac activation, cell adhesion, and protrusion, through a novel mechanism regulated by FAs [39].

The phosphorylation of serine residues at paxillin LIM domains has been suggested to regulate paxillin localization at FAs, as well as cell adhesion to fibronectin. Integrin binding to ECM promotes the phosphorylation of Ser188 and Ser190, whereas Ser112, 173, 217, and 259 at LD domains and Ser501 at LIM3 are substrates for protein kinase A (PKA). Also, Ser residues 85, 90, 94, 106, 108, 126, and Ser227 at LD3 domain are phosphorylated by GSK3, whereas Akt posphorylates Ser173. The serine residues 398 and 403 at LIM1, 457 at LIM2, and 481 at LIM3 are phosphorylated not only as a consequence of cell adhesion but also by angiotensin II stimulation. Additionally, protein kinase C (PKC) has been found to phosphorylate serine residues 91, 98, 108, 382, and Thr 295 and 540. Moreover, ERK 1/2 which localize to FAs and regulate paxillin disassembly from adhesion complexes, phosphorylate paxillin at Ser106, 231, and 290 [40, 41].

Although the precise involvement of serine phosphorylation in the regulation of adhesion dynamics and migration is not fully understood, the phosphorylation of Ser272 at the LD4 motif seems to regulate the nuclear-cytoplasmic localization of paxillin by blocking the nuclear export of paxillin, thus allowing nuclear accumulation. Recent evidence on this matter shows that nuclear paxillin promotes DNA synthesis and proliferation, and also suppresses H19 gene expression, which is a parental imprinting gene hypothesized to be a tumor supressor [5].

Paxillin incorporation to FAs is conformationally regulated and is essential for the recruitment of FAK and other scaffolding and signaling molecules involved in FA assembly. The autophosphorylation of FAK at Tyr397 upon integrin activation generates a binding site for the SH2 domain of Src tyrosine kinases, thereby recruiting Src to FAs for the formation of an active FAK/Src kinase complex which phosphorylates distinct focal adhesion proteins including FAK, paxillin and p130cas, leading to the activation of signaling to cell migration [30]. In addition to Src recruitment, FAK has been shown to transmit integrin-stimulated signals to JNK. Recently, paxillin Ser178 has been identified as a major target for JNK, and proven essential for integrin-induced cell movement by means of the JNK–paxillin connection. The activation of JNK by the association of the small GTPase Rac with ERK1/2 and the downstream phosphorylation of paxillin at Ser178 have been proven essential for distinct stages of cell movement and migration [37]. Paxillin phosphorylation also allows binding to the SH2/SH3 domains of CRK, involved in cell mobility through the activation of Rac [29]. On this line, phospho-paxillin binding to the SH2 domain of p120RasGAP results in the activation of p190RhoGAP and the consequent decrease in the activity of RhoA, which could allow cell movement through the disassembly of the actin cytoskeleton [51].

In addition to integrins, a diversity of stimuli including bombesin, lysophosphatidic acid, and PDGF promote paxillin tyrosine phosphorylation by FAK at FAs [35, 42]. Whereas the phosphorylation of paxillin at Tyr118 regulates the affinity of FAK binding to paxillin, the dephosphorylation of phospho-Tyr118 may retard cell migration even when accompanied by increased expression of phospho-FAK Tyr397, which further underlines the importance of paxillin Tyr118 phosphorylation in the full activation of the FAK/paxillin complex and the induction of cell migration.

Of notice, paxillin isoforms exhibit important differences regarding FAK and vinculin binding. Compared to paxillin α, isoform β exhibits reduced in vitro binding to vinculin but equivalent binding to FAK. In contrast, Paxillin γ is defective for FAK binding in vitro but retains vinculin-binding activity. The reason for these differences is not clear, since the insertions do not appear to disrupt the LD4 motif, which binds FAK/vinculin. It is possible that the insertions disrupt interactions with binding partners that stabilize the association of FAK or vinculin with paxillin. Alternatively, these insertions may alter the conformation of the protein masking recognition of both the LD2 and LD4 motifs by FAK or vinculin [11].

Paxillin interacts with vinculin at FAs through the C-terminal region of vinculin which functions as a binding site for the LD1, LD2, and LD4 domains of paxillin. The high degree of homology of the FAT region of FAK with the C-terminal tail of vinculin suggests that vinculin oligomers could simultaneously bind the LD2 and LD4 motifs, thus interfering with FAK binding to paxillin [52, 53]. Not only vinculin phosphorylation at Tyr1065 regulates its conversion to an open ligand conformation thus allowing binding to talin, F-actin, and other cytoskeletal-related proteins, but it has also been involved in the regulation of apoptosis mediated by the ERK1/2 pathway [54]. Hence, although vinculin and paxillin colocalize at focal adhesions, these two proteins function independently, and vinculin could subserve a broader role in cell cytoskeletal dynamics [55, 56].

Polarized cell migration is essential for normal organism development and is also a critical component of cancer cell invasion and disease progression. Directional cell motility requires the coordination of dynamic cell–ECM interactions as well as repositioning of the Golgi apparatus, both of which are controlled by the microtubule (MT) cytoskeleton. Dynamic microtubules closely interact with FAs in distinct migrating cells. Examination of microtubule dynamics associated with FAs showed that paxillin acts as an upstream regulator of site-specific microtubule transition from growth to shrinkage (“catastrophe”) at specific, paxillin-rich adhesion domains. Tubulin binds to paxillin LIM2 and LIM3 domains, which requires the integrity of the paxillin zinc finger sequences. Furthermore, replacement of full-length paxillin at adhesion sites by microinjected paxillin LIM2-LIM3 domains suppressed “catastrophe” exclusively at adhesions, suggesting that paxillin regulates microtubule dynamics at adhesion complexes [57].

In addition to its role in FAs, paxillin has been linked to distinct cellular processes in cells lacking these adhesions. In hematopoietic T cells, paxillin is constitutively associated with microtubules and microtubule-organizing centers (MTOC) in a phosphorylation-independent manner. Moreover, paxillin association with the peripheral supramolecular activation center (pSMAC) contributes to the reorganization, docking, and specific location of MTOCs. Along this line, the Src family members Lck (lymphocyte-specific protein tyrosine kinase) and Fyn are key upstream mediators of PI3K and MAPK signaling pathways. As a downstream target of these tyrosine kinases, paxillin has been proposed to contribute to the reorientation and function of the MTOC [24] by recruiting Pyk2 to the MTOC [58]. Paxillin has also been shown to interact with the N- and the C-terminus of γ-tubulin in T lymphocytes, suggesting that paxillin could be responsible for linking microtubule extremes to filamentous actin cortical termini [59].

Recent work has identified a new and conserved role for paxillin in the regulation of the post-translational modification of the MT cytoskeleton, through an inhibitory interaction with the α-tubulin deacetylase HDAC6, mediated by the proline-rich domain of paxillin. Through this interaction, paxillin regulates Golgi organelle integrity as well as polarized cell invasion and migration in normal and transformed cells [60]. Since FAK-mediated activation of RhoA has been shown to stimulate MT acetylation [61], it is plausible that RhoA activation may promote the association of paxillin and HDAC6 and thus result in HDAC6 inhibition and concomitant enhancement of MT acetylation.

The innate immune function of phagocytosis of apoptotic cells, tissue debris, pathogens, and cancer cells is carried out by resident microglia in the central nervous system (CNS). In healthy cells, this process is arrested by the recruitment of immune inhibitory receptor SIRPα to the surface of phagocytes and the resulting activation of signals that inhibit phagocytosis. Recent work showed that the activation of SIRPα prevents paxillin phosphorylation indirectly, through the inactivation of cofilin, induced by paxillin dephosphorylation at Tyr118. These observations support a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by phagocytic receptor signaling to the activation of paxillin and cofilin, and signaling by immune inhibitory SIRPα which promotes the inactivation of paxillin and cofilin [81].

Blood leukocytes are involved in essential functions including the development of the immune system and the control of the inflammatory response, which requires migration of leukocytes and lymphocytes from the vasculature, controlled by integrin receptor signaling. Within this process, paxillin binding to α4 integrin cytoplasmic domain induces ligation of integrin α4β1, thus promoting the signaling events. Since paxillin can associate with nonreceptor tyrosine kinases such as FAK and Pyk2, signaling from integrins to these kinases may be implicated in the regulation of leukocyte migration [82, 83].

In inflammatory responses, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessels to the site of injury or infection. Recent findings showed a selective loss of paxillin and FAK from FAs in proximity to transmigrating neutrophils in HUVEC endothelial cells. As paxillin dynamics are partially regulated by FAK, decreases in FAK protein or disruption of FAK signaling prevents neutrophil transmigration, revealing a novel role for endothelial FA proteins paxillin and FAK in the regulation of inflammation and immune responses [84].

Paxillin serves as an ERK-regulated scaffold for coordinating FAK and Rac activation, essential for cytoskeleton rearrangement involved in epithelial morphogenesis [40, 85]. The regulation of FA formation/turnover and the interaction of FAs with the actin cytoskeleton are central events for the morphogenic actions of distinct growth factors. Among them, hepatocyte growth factor (HGF) stimulation promotes Src activation and the recruitment of inactive ERK to paxillin, which induces ERK activation. The phosphorylation of paxillin by activated ERK enhances FAK recruitment to nascent adhesions. Ser83 has been identified as the ERK phosphorylation site in murine paxillin, which is essential for HGF-stimulated paxillin–FAK association, FAK phosphorylation, PI3K activation, and the subsequent activation of Rac [47]. This process allows the disassembly of specific adhesions surrounding HGF receptor along morphogenesis, thus promoting lamellipodia formation, cell spreading, and migration [41].

Signaling through integrin receptors is importantly involved in developmental processes mainly related to mesodermal-derived structures. Although evidence regarding paxillin role in development is scarce, as a well-known integrin effector, paxillin acts as transducer of fibronectin receptor signals during early development by promoting the recruitment and phosphorylation of FAK, p130cas, and MAPKs [86]. Thus, paxillin and fibronectin seem to regulate some common embryonic developmental events, by controlling FA dynamics, cell migration, and spreading [53, 87]. In support of this assumption, the deletion of the paxillin gene in mouse embryos is lethal. Specifically, phosphorylation of paxillin by PAK (kinase activated by p21) at Ser273 in the chick (Ser272 in human) has been shown to promote the adhesion and migration of non-neural cells [88]. Also, in this line, the activity of dipeptidyl peptidase IV family protease (DPP9) has been shown to be essential for mice neonatal survival. DPP9 gene silencing or specific inhibition was shown to reduce cell adhesion and migration required for early embryonic development by decreasing the phosphorylation of FAK and paxillin [89]. In fact, the death of paxillin null mice embryos after gastrulation derives from defective mesodermal development [77].

Evidence has been provided demonstrating FAK and paxillin involvement in the regulation of cytoskeletal changes fundamental to neuronal development, such as the hormonal modulation of FAK and paxillin in the feminization of the brain. Whereas FAK and paxillin are highly expressed in the hypothalamus of female rats at birth, the male tissue shows low expression, which is significantly increased by the inhibition of aromatase, suggesting that the combined action of androgens and estrogens may be important in paxillin regulation along development [90].

Integrin-dependent functions, such as adhesion, migration, and proliferation, are critical in the development and the maintenance of the central nervous system [91]. Integrin-activated signaling, including paxillin phosphorylation, is required for the outgrowth of neurites and the formation of the glial scaffold in the brain and also for remodeling and long-term potentiation within synapses. Paxillin involvement in focal adhesion dynamics plays an important role in neurite extension, and is also required for neurite outgrowth, although the molecular mechanism by which paxillin may regulate these processes is poorly understood [92]. Recent studies have shown that neurite outgrowth induced by laminin requires the phosphorylation of FAK and paxillin, mediated by AKT/GSK-3β signaling. This process seems to be essential for brain development, since the knock-out of laminin induces the decrease of FAK and paxillin phosphorylation accompanied by severe anatomic and neuronal morphological defects [93]. In addition to serine phosphorylation, tyrosine phosphorylation of paxillin has also been shown to promote neurite outgrowth induced by NGF and EGF-dependent pathways. Additionally, Nemo-like kinase has been suggested to control the dynamics of the cytoskeleton downstream of NGF signaling, through directly phosphorylating microtubule-associated protein-1B (MAP1B) and paxillin at Ser126 [94]. Furthermore, p38MAPK-mediated phosphorylation of Ser85 on paxillin is also involved in NGF-induced neurite outgrowth in PC-12 cells, since mutation of paxillin Ser 85 significantly inhibits NGF-induced neurite extension. Hence, the phosphorylation of paxillin at Tyr and Ser residues is required for NGF-induced neurite extension, probably through regulating focal adhesion organization [95].

Radial glial cells are known to support neuronal migration along the development of the nervous system. Although little is known regarding the role of paxillin in this process, phosphorylation of paxillin at Ser178 has been shown to play a key role in cell migration in the central and peripheral nervous systems. During development of the peripheral nervous system (PNS), intercellular signaling between neurons and Schwann cells induces the migration of Schwann glial cells along axons, wrapping individual axons to form a myelin sheath. In spite of the considerable clinical importance of myelination, the molecular mechanisms underlying this process in the peripheral nervous system as well as within the central nervous system is largely undefined. Recent data on this issue have shown that JNK binds to the first LD region of paxillin and phosphorylates Ser178 to regulate Schwann cell migration, illustrating that paxillin provides one of the convergent points of intracellular signaling pathways controlling neuronal-mediated Schwann cell migration [96].

Oligodendrocytes (OLs) are the myelinating glial cells of the central nervous system (CNS). Oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes (OLs) in order to form the myelin sheath, which wraps axons and allows the rapid propagation of action potentials in the vertebrate nervous system. Studies from Miyamoto et al. [97] have recently shown that cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase enriched in neuronal tissues, directly phosphorylates paxillin at Ser244 following the induction of oligodendrocyte differentiation. Since oligodendrocyte differentiation and myelin sheath formation are prevented by the suppression of Cdk5, paxillin phosphorylation by Cdk5 seems to be a key mechanism in OL differentiation and may ultimately regulate myelination.

Conditions involving oxidation, mechanical traction, or hypotonic challenge (HTS) have been shown to induce FAK and paxillin phosphorylation and the translocation/activation of RhoA. Athough FAK and paxillin tyrosine phosphorylation is not required for the activation of RhoA/RhoK signaling, the simultaneous activation of RhoA/Rho kinases and FAK/paxillin, driven either by mechanical stress or by receptor activation-induced ATP release, has been found to promote actin cytoskeleton reorganization leading to functional repercussion [98].

Oxidative stress is one of the most common apoptosis-inducing factors in several organs, from the intestinal and cardiovascular systems to neuronal cells, including the sensory organs [99]. In pathological events involving ischemia–reperfusion, reactive oxygen species (ROS), such as superoxide anion (O2·2), hydrogen peroxide (H₂O₂), and hydroxyl radical (HO·), are released by the ischemic tissue. ROS, particularly the hydroxyl radical (HO·), are capable of stimulating the tyrosine phosphorylation of paxillin, FAK, and p130cas in human endothelial cells which, in turn, triggers the activation of signaling cascades that regulate cell proliferation and differentiation. Importantly, recent studies show that PAX-FAK-p130cas phosphorylation induced by intracellular hydroxyl radical (HO·) stimulates polymorphonuclear lymphocyte (PMN) adhesion to human endothelial cells and their subsequent migration, suggesting an important role of FAK/PAX activation by ROS in PMN recruitment during inflammation [100].

The regulation of paxillin phosphorylation seems to be a target for the control of endothelial cell (EC) barrier function and its dysfunction induced by oxidative stress, as well as lipopolysaccharide- and ventilator-induced lung injury. The release of oxidized phospholipids (OxPLs) into the circulation as a consequence of tissue injury has not only been shown to contribute to pulmonary endothelial cell dysfunction and the alteration of vascular barrier function but can also play a barrier-protective role in human lung ECs through Rac- and Cdc42-dependent promotion of actin reorganization and paxillin accumulation at focal adhesions. Moreover, phosphorylation of paxillin at Ser273 by PAK1 is critically involved in the positive feedback regulation of the Rac-PAK1 pathway, which may contribute to the sustained enhancement of the EC barrier induced by OxPLs [101]. In contrast, paxillin Tyr31 and Tyr118 phosphorylation has been implicated in EC barrier dysfunction causd by OxPL, through the destabilization of VE-cadherin at adherens junctions [1]. Although the opening of endothelial cell junctions causes increased permeability, ECs are capable of enhancing barrier function in the presence of barrier-enhancing factors such as sphingosine-1-phosphate (S1P), hepatocyte growth factor (HGF), and hyperosmolality. This process is driven by HGF- and S1P-dependent modulation of Rac–Rho signaling which, in turn, requires c-Src/FAK-mediated phosphorylation of paxillin at Tyr31 and Tyr118 and also at Tyr181 by c-Abl kinase [102, 103].

Paxillin involvement in cell migration was initially suggested by the high levels of expression of the phosphorylated protein determined in several cancer tissues and metastatic cancer cells, in parallel to increased epithelial-mesenchymal transition [104].

A main characteristic of transformed epithelial cancer cells is the ability to survive and proliferate in the absence of contact with immobilized ECM [105]. Paxillin is known to acquire gain of function mutations that are associated with alterations in the malignant progression of many tumors including breast, lung, prostate, melanoma, and colorectal cancer [106]. Among these, glioblastoma multiforme (GBM), the most common and malignant type of glioma, remains one of the most lethal cancers in the central nervous system (CNS). Recent studies have demonstrated that the overexpression of paxillin at the RNA and protein level is associated with GBM tumor malignancy and hence, predictive of poor survival [107]. These data identify paxillin as a novel prognostic biomarker with potential anti-invasion therapeutic implications in GBM. On this line, a total of 21 unique paxillin mutations were identified in lung cancer tissue specimens and cell lines. A127T, the most common of these mutations, increases tumor growth and invasion in vivo [108]. Importantly, paxillin δ is able to suppress full-length (α)-paxillin signaling as well as interactions with actin-binding and integrin-linked proteins due to the lack of LD1 domain, thus suggesting a potential role in suppressing the migratory phenotype of cancer cells [10].

Recent data show that maximal tyrosine phosphorylation of paxillin by Src and FAK is required for the induction of anchorage-independent signal transduction and proliferation, characteristic of metastatic cells [105, 109]. Along this line, studies by Sero et al. [110] in embryonic fibroblasts (MEF) show that paxillin integrates physical and chemical motility signals by spatially constraining the location of motile processes, thereby regulating directional migration. These findings indicate that complex multimeric and competing interactions on paxillin are required to augment anchorage-independent cell proliferation in cancer development.

A growing number of signaling molecules have been shown to promote or regulate cell migration of cancer cells through the phosphorylation of paxillin at Tyr118 and Ser178 by FAK, which alters the organization of FAs, with the consequent promotion of cell motility [2, 111]. In fact, the increased phosphorylation of these residues is considered as indicative of metastasis.

Multiple receptor-activated signaling pathways have been involved in cell transformation and migration underlying metastasis in breast cancers. Particularly, paxillin has been shown to control the signaling of estrogen (17-β estradiol) to FAK/N-WASP/Arp2/3 complex in breast cancer cells [112]. On this line, the vitamin A derivative retinoic acid (RA), frequently used in cancer therapy, has been shown to induce Src/FAK/PI3K complex signaling to cell attachment, migration, and invasion mediated by the rapid activation of the actin-binding protein moesin. Within this pathway, cell migration of breast cancer cells is prevented by RA through the regulation of the expression of Moesin and the downstream inhibition of Src, FAK, and paxillin activity, providing novel mechanistic clues for the development of new drugs for cancer treatment [113]. Also on this matter, paxillin has been shown to be transcriptionally upregulated and phosphorylated by human epidermal growth factor receptor-2 (HER2) signaling in vitro. Since HER2 overexpression has been involved in metastatic cancer progression, particularly in breast cancer, paxillin has been identified as a marker of aggressive breast cancer and a promoter of neoplastic growth and invasion, supporting its role as a biomarker or therapeutic target [114, 115].

Rho/ROCK signaling plays a crucial role in the regulation of FAs and cell motility [116]. The serine/threonine protein phosphatase inhibitor calyculin A has been shown to promote focal adhesion assembly upon the inhibition of the ROCK downstream target serine/threonine myosin light chain (MLC) phosphatase, and the resulting tyrosine phosphorylation of FAK, paxillin, and p130cas, thus supporting calyculins as potential anticancer agents [117]. The FAK/paxillin pathway regulates small Rho GTPases including RhoA, Rac1, and Cdc42, which are critical determinants of cancer cell migration. Importantly, the specific interaction of Rho GTPases with tumor supressor deleted in liver cancer-1 (DLC1) has been shown to decrease FAK-dependent localization of paxillin at immature focal adhesions, thus controlling the lifetime of nascent focal adhesions [118]. Since this process is FAK-independent in migrated cells, these data indicate that the function of paxillin/FAK/RhoA signaling in the control of cell movement is developmentally regulated. On this matter, the serine/threonine kinase maternal embryonic leucine zipper kinase (MELK) has recently been shown to promote cell migration and invasion via the activation of RhoA and the downstream phosphorylation of FAK and paxillin. Hence, MELK has emerged as a potential cancer biomarker [119]. The human phosphatase and tensin homolog (PTEN) tumor suppressor is mutated at high frequency in a large number of cancers. Recent work in colon cancer cells has shown that PTEN function as a tumor suppressor derives from the downregulation of paxillin expression by the inhibition of PI3K/AKT/NF-kB signaling. This allows the activation of NF-kB and the promotion of paxillin expression, thus stimulating cell invasion, migration, and cancer progression [14]. In agreement with this evidence, proteolysis of paxillin by calpain between the LD1 and LD2 motifs in HeLa cells generates a carboxy-terminal 55 KDa fragment, similar to delta paxillin, which functions as an antagonist of endogenous paxillin and may function to limit cancer cell invasion by preventing the assembly/disassembly of FAs and cell migration [120, 121].

Recent studies in human pancreatic ductal adenocarcinoma cells (PANC-1) have shown that the elevation of cAMP concentration by distinct stimuli results in the inhibition of migration of several PANC cell types in strict correlation with the inhibition of FA turnover, a highly significant loss of paxillin from FAs, and the consequent cessation of ruffling. These data clearly indicate that the inhibitory effect of cAMP on migration, ruffling, FA dynamics, and paxillin localization is mediated by the downstream activation of PKA, while the inhibition of this enzyme potentiates migration [122].

Hepatocyte growth factor (HGF)-induced c-Met signaling plays critical roles in the progression of hepatocellular carcinoma (HCC). On this line, PKCε-mediated c-Met endosomal processing was shown to stimulate c-Met-JNK-induced paxillin (Ser178) phosphorylation, which is required for cell migration, invasion, and intrahepatic metastasis [123].