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Regulation of cell-matrix adhesion dynamics and Rac-1 by integrin linked kinase

Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR6543, Centre Antoine Lacassagne, Nice, France

¹Correspondence: Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR6543, Centre Antoine Lacassagne, 33 Ave. de Valombrose, Nice 06189, France. E-mail: vanobber{at}unice.fr

SPECIFIC AIMS

Extracellular matrix (ECM) receptors of the integrin family initiate changes in cell shape and motility by triggering the formation of large multiprotein complexes beneath the membrane, referred to as cell-matrix adhesions. These complexes physically link integrins to the actin cytoskeleton and functionally couple them to the appropriate intracellular signaling networks. Integrin-linked kinase (ILK) is a widely expressed partner of ßbeta;1 integrins that participates in dynamic rearrangement of cell-matrix adhesions and cell spreading by mechanisms that are not well understood. To further delineate the mechanism by which ILK regulates these events, we used a gain-of-function strategy by targeting ILK to the plasma membrane.

PRINCIPAL FINDINGS

1. Membrane-targeted ILK increases cell spreading and modifies cell-matrix adhesion dynamics in fibroblasts
A plasma membrane-anchored form of ILK was engineered by fusing its C-terminus to a green fluorescent protein (GFP) fitted with the farnesylation sequence of H-Ras (ILK-GFP-F) (Fig. 1 A–C). The most striking phenotype of fibroblasts stably expressing this chimera was their increased size (1.8-fold larger than control ILK-GFP cells, Fig. 1D ), suggesting that membrane targeting alone is able to activate the cell machinery involved in cell spreading.

View larger version (32K): [in this window] [in a new window]   Figure 1. Membrane-targeted ILK. A) Schematic representation of membrane-targeted integrin-linked kinase (ILK-GFP-F). The C-terminal of ILK was fused to a green fluorescent protein (GFP) fitted with the farnesylation sequence of H-Ras. B) ILK-GFP and ILK-GFP-F expression in CCL39 fibroblasts was monitored by Western blotting using an anti-ILK Ab (DI). C) Exponentially growing ILK-GFP and ILK-GFP-F cells were subjected to hypotonic lysis and isolation of the membrane (M) and soluble (S) fractions was performed. Ten micrograms of protein were separated by SDS-PAGE and analyzed by Western blotting using antitransferrin receptor as a marker for the membrane and anticaspase 9 as a marker for the cytosol. Results are representative of 2 independent experiments. Chimeric ILK proteins were detected with anti-V5 Ab, and endogenous ILK was detected with the DI anti-ILK Ab. D) The mean surface area (±SEM, n>60) was determined on F-actin-stained cells 20 h after plating on fibronectin-coated coverslips. E) CCL39-derived cells expressing EGFP-F, ILK-GFP, or ILK-GFP-F were plated on fibronectin-coated coverslips for 20 h then fixed, permeabilized and stained for paxillin. Digital overlay of images was performed using MetaMorph software. Scale bar = 20 µm.

ILK-GFP-F was found to be highly enriched cell-matrix adhesions (Fig. 1E ). Membrane targeting of ILK induced a significant increase in formation of focal complexes and their maturation into longer, more mature adhesions. These elongated adhesions do not fulfill the molecular requirements for classical fibrillar adhesions and are likely to represent elongated focal adhesions undergoing some maturation process. Paxillin staining was enriched in the same cell-matrix adhesions as ILK-GFP-F (Fig. 1E ), suggesting that ILK localization can direct paxillin recruitment to cell-matrix adhesions, whereas the inverse is usually thought to be the case.

2. Membrane-targeted ILK increases Rac-1 activation
In light of the increased cell spreading observed in ILK-GFP-F cells, and the known role for Rac in this process, the ability of ILK-GFP-F to control Rac-1 activity was assessed in pull-down assays using glutathione S-transferase (GST)-CRIB (PAK3) to sequester GTP-bound Rac in lysates of cells expressing ILK-GFP or ILK-GFP-F. Rac-1 activation was markedly higher in cells expressing the membrane-anchored chimera, and this was observed in both fibroblasts (Fig. 2 ) and endothelial cells. In agreement with a role for ILK in Rac activation, ILK silencing decreased cell spreading and Rac-1-GTP levels in endothelial cells.

View larger version (24K): [in this window] [in a new window]   Figure 2. Membrane-targeted ILK increases Rac-1 activity in CCL39 fibroblasts. Activation of Rac-1 was measured in a GST-CRIB (PAK3) pull-down assay on (A) exponentially growing ILK-GFP and ILK-GFP-F expressing attached cells (Att), cells maintained in suspension in serum-free medium for 15 min (Sus) or cells plated on fibronectin-coated dishes (10 µg/ml) for 10 min (FN). B) Active Rac-1 was measured in CCL39 fibroblasts expressing ILK-GFP-F under control of a tetracycline-sensitive promoter in serum-deprived nontreated (control) or tetracycline-induced cells (1 µg/ml, 20 h) (+Tet) after adhesion (10 min) to fibronectin (FN) or stimulation or adherent cells with 10 ng/ml platelet-derived growth factor. ILK-GFP-F expression was monitored by Western blot analysis using anti-V5 Ab (left panel). Immunoblots were quantified and results were expressed as fold increase relative to control. Results represent mean values (±SEM) from 3 independent experiments.

Interestingly, constitutively active Rac (RacV12) expression only partially rescued the spreading defect of ILK-deficient cells, as compared to control cells expressing the constitutively active GTPase, indicating that ILK can regulate cell spreading both through Rac-1 activation and another unidentified pathway, possibly involving the turnover of cell-matrix adhesions.

3. Role for PKL and ßbeta;PIX in ILK-dependent activation of Rac-1
To identify a molecular link between ILK and Rac-1 activation, we examined the role of ßbeta;PIX, a Rac/Cdc42 guanine nucleotide exchange factor (GEF) of the COOL/PIX family since it has previously been shown that the ILK partner, paxillin, can bind directly to the Arf-GTPase activating protein proteins GIT1 and GIT2/PKL. PKL, in turn, binds to and activates the Rac/Cdc42 GEF, ßbeta;PIX. Endogenous ILK coimmunoprecipitated with both PKL and ßbeta;PIX suggesting that they may associate in a functional complex in intact cells. When ILK-GFP-F was coexpressed with the DH-ßbeta;PIX mutant, Rac-1 activity decreased by a factor of 2. These results indicate that the ßbeta;PIX, likely via the ILK/PKL/ßbeta;PIX complex, participates in Rac-1 regulation by ILK.

CONCLUSIONS AND SIGNIFICANCE

Using a novel gain of function strategy (a membrane-targeted fluorescent chimera of ILK) and a loss of function approach (siRNA) we show here in fibroblasts and endothelial cells that plasma membrane targeting of ILK is sufficient to direct its recruitment to cell-matrix adhesions. ILK-GFP-F expression increases the size and density of integrin-based adhesions by enhancing their formation as well as their stability. In addition, ILK-GFP-F promotes cell spreading on fibronectin and induces a constitutive increase in the levels of GTP-bound Rac-1 by a mechanism involving ßbeta;PIX. Whereas ILK-dependent activation of Rac-1 is an important event in integrin-mediated regulation of the actin cytoskeleton and cell morphology, our findings that constitutively active Rac expression only partially restores the spreading defects of ILK-depleted cells suggest that an additional ILK-dependent signal, likely to involve the turnover of cell-matrix is adhesions, is required for cell spreading (Fig. 3 ). Altogether, these findings advance our current knowledge of adhesion signaling by providing new mechanistic insights into the role of ILK in coordinating cellular responses to the ECM.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the regulation of cell-matrix adhesion dynamics and cell spreading as determined by expressing a constitutively membrane-anchored form of ILK (ILK-GFP-F) or ILK silencing by RNA interference. ILK-GFP-F is retained in integrin-based adhesive structures, where it enhances their formation and stability. The ILK/PKL/ßbeta;PIX complex participates in Rac-1 regulation by ILK. Regulation of cell spreading by ILK proceeds through Rac-dependent and Rac-independent mechanisms.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4579fje

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