Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1beta signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.
"SHP-2, a non-receptor tyrosine phosphatase, translocates from the ER to focal adhesions in response to src signaling [Wang et al., 2006] to dephosphorylate FAK on Y 925 and paxillin on Y 31 , thus promoting detachment from the extracellular matrix [Vadlamudi et al., 2002]. Like CaMK-II inhibition, the knockout or inhibition of SHP-2 increases focal adhesion size and blocks cell motility in both cells in culture and in mouse embryos [Yu et al., 1998; Manes et al., 1999; Inagaki et al., 2000; Saxton et al., 2000; MacGillivray et al., 2003; Wang et al., 2005]. While SHP-2 activity is known to be regulated by tyrosine phosphorylation, this enzyme can also be phosphorylated on serine/threonine residues [Poole and Jones, 2005]. "
[Show abstract][Hide abstract] ABSTRACT: Transient elevations in Ca2+ have previously been shown to promote focal adhesion disassembly and cell motility through an unknown mechanism. In this study, evidence is provided to show that CaMK-II, a Ca2+/calmodulin dependent protein kinase, influences fibroblast adhesion and motility. TIRF microscopy reveals a dynamic population of CaMK-II at the cell surface in migrating cells. Inhibition of CaMK-II with two mechanistically distinct, membrane permeant inhibitors (KN-93 and myr-AIP) freezes lamellipodial dynamics, accelerates spreading on fibronectin, enlarges paxillin-containing focal adhesions and blocks cell motility. In contrast, constitutively active CaMK-II is not found at the cell surface, reduces cell attachment, eliminates paxillin from focal adhesions and decreases the phospho-tyrosine levels of both FAK and paxillin; all of these events can be reversed with myr-AIP. Thus, both CaMK-II inhibition and constitutive activation block cell motility through over-stabilization or destabilization of focal adhesions, respectively. Coupled with the existence of transient Ca2+ elevations and a dynamic CaMK-II population, these findings provide the first direct evidence that CaMK-II enables cell motility by transiently and locally stimulating tyrosine dephosphorylation of focal adhesion proteins to promote focal adhesion turnover.
Cell Motility and the Cytoskeleton 08/2008; 65(8):662-74. DOI:10.1002/cm.20294 · 4.19 Impact Factor
"). SHP-2, which is encoded by PTPN-11, is a vital adaptor protein located downstream of growth factor/ras signalling (Cai et al., 2002; Shi et al., 2002). Activated SHP-2 induces activation of downstream target molecules, including Erk1/2 (Cai et al., 2002; MacGillivray et al., 2003). In haematopoietic cells, SHP-2 positively regulates IL-3-induced activation of Jak2 kinase through a mechanism requiring its catalytic activity (Berchtold et al., 1998; Yu et al., 2003). "
[Show abstract][Hide abstract] ABSTRACT: The Helicobacter pylori virulence factor, CagA, is causally linked to lymphoma of gastric mucosa-associated lymphoid tissue (MALT). However, it is unclear how CagA promotes the development of gastric MALT lymphoma. We investigated whether CagA modulates the activation of Erk1/2 and their downstream apoptosis regulators in B lymphocytes. Transfection of B1 lymphocytes with cagA transiently increased Erk1/2 phosphorylation, which was negatively regulated by MKP-1 and MKP-6. Activation of Erk1/2 led to phosphorylation of Bad at Ser-112, as confirmed with a chemical Erk1/2 inhibitor. However, CagA-induced Erk1/2 activation did not alter expression of either Bcl-2 or Bax. Importantly, cagA-transfected B1 cells were significantly protected against apoptosis induced by hydroxyurea. Our results reveal that CagA, to some extent like IL-3, can enhance lymphocytes' ability to evade apoptosis through phosphorylation of Bad. This may account, at least in part, for the ability of CagA to promote lymphomagenesis.
"Multiple adhesive contacts with ECM ligands depend on recruitment of actin-binding proteins into focal complexes that are associated with the termini of actin-containing stress fibres that communicate extracellularly via integrins. Such complexes can be demonstrated by physically extracting, then identifying the proteins that bind to ligand-coated beads (Plopper and Ingber, 1993; Glogauer et al., 1997; MacGillivray et al., 2003). In this study, the recovery of both immunoreactive b 1 integrins and b-actin that were extracted with collagencoated beads from Msp-and control vehicle-treated cells was equivalent, suggesting that avidity through the formation of integrin–actin complexes was not affected by exposure to Msp. "
[Show abstract][Hide abstract] ABSTRACT: Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.
Aretha Fiebig, Julien Herrou, Jonathan Willett, Sean Crosson
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.