Herreros, L. et al. Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton. J. Biol. Chem. 275, 26436-26440

Complutense University of Madrid, Madrid, Madrid, Spain
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2000; 275(34):26436-40. DOI: 10.1074/jbc.M003970200
Source: PubMed


Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural
and signaling molecules. α-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal
LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of α-tubulin with the C terminus of paxillin. Another member of the tubulin
family, γ-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both α- and γ-tubulin
in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent
T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy
revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with α-
and γ-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to
integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton,
and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the
lymphocyte microtubule network.

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    • "Paxillin serves as an adapter protein, mediating signal transduction from the extracellular matrix to focal adhesions and the actin cytoskeleton [11], [19]. Previous studies showed that C-terminal LIM domains in paxillin are involved in binding the protein tyrosine phosphatase PTP-PEST to target the protein to focal adhesions, and also to bind α- and γ-tubulin to direct an interplay between actin filaments and microtubules [20]–[22]. Through its LD motifs at N-termini, paxillin interacts with actopaxin (a member of the parvin family of focal-adhesion proteins), ILK (integrin-linked kinase), FAK (focal adhesion kinase), PKL (paxillin kinase linker) and vinculin to regulate Rho GTPase signaling and focal adhesion turnover [20], [21], [23], [24]. However, no LD motif has been discovered in the paxillin equivalent of yeasts and filamentous fungi, and only two or three LIM domains are present [25], [26]. "
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    ABSTRACT: LIM domain proteins contain contiguous double-zinc finger domains and play important roles in cytoskeletal re-organisation and organ development in multi-cellular eukaryotes. Here, we report the characterization of four genes encoding LIM proteins in the rice blast fungus Magnaporthe oryzae. Targeted gene replacement of either the paxillin-encoding gene, PAX1, or LRG1 resulted in a significant reduction in hyphal growth and loss of pathogenicity, while deletion of RGA1 caused defects in conidiogenesis and appressorium development. A fourth LIM domain gene, LDP1, was not required for infection-associated development by M. oryzae. Live cell imaging revealed that Lrg1-GFP and Rga1-GFP both localize to septal pores, while Pax1-GFP is present in the cytoplasm. To explore the function of individual LIM domains, we carried out systematic deletion of each LIM domain, which revealed the importance of the Lrg1-LIM2 and Lrg1-RhoGAP domains for Lrg1 function and overlapping functions of the three LIM domains of Pax1. Interestingly, deletion of either PAX1 or LRG1 led to decreased sensitivity to cell wall-perturbing agents, such as Congo Red and SDS (sodium dodecyl sulfate). qRT-PCR analysis demonstrated the importance of both Lrg1 and Pax1 to regulation of genes associated with cell wall biogenesis. When considered together, our results indicate that LIM domain proteins are key regulators of infection-associated morphogenesis by the rice blast fungus.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "Shoc2 is a critical modulator of the ERK1/2 pathway and was first identified in C. elegans (named SOC-2/SUR-8) [18]–[20]. Shoc2 forms a ternary complex with Ras and Raf-1 proteins [21], thereby positively regulating Ras-mediated signaling [19]. More recently, it was demonstrated that Shoc2 regulates ERK1/2 activity as a part of a holoenzyme comprised of Shoc2 and the catalytic subunit of protein phosphatase 1c (PP1c) [22]. "
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    ABSTRACT: Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.
    Full-text · Article · Jun 2013 · PLoS ONE
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    • "The paxillin N-terminus contains several short alpha-helical motifs that bind other FA proteins and signaling molecules, such as vinculin [24], FAK and Src kinases [43] and the Arf6GAP PKL [41], as well as a polyproline domain and long regions of random coil [44]. The C-terminus is composed of four tandem LIM zinc finger domains, and it contains the FA-targeting sequence [45], as well as binding sites for tubulin [46] and and PTP-PEST [47], [48], [49]. Importantly, calpain-mediated proteolysis of paxillin regulates lamellipodia formation [50] and FA dynamics [51], suggesting that the N- and C-termini might also have distinct effects on cell physiology. "
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    ABSTRACT: Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5-10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax-/- and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax-/- cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax-/- and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.
    Full-text · Article · Dec 2011 · PLoS ONE
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