Philippe Rondé

Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397

One of the major tyrosine phosphorylation activities linked to integrin signalling is that of focal adhesion kinase (FAK). High amounts of FAK are located at specialised subcellular compartments known as focal adhesions. FAK tyrosine phosphorylation at focal adhesions is increased by various stimuli including integrin engagement during migration processes, growth factors and oncogene transformation. Phosphorylation of FAK at various tyrosine residues regulates focal adhesion turnover by mechanisms that are not well understood. We made a fluorescent FAK mutant (Y397F-FAK/YCam) to analyse, in living cells, how phosphorylation of FAK regulates the turnover of focal adhesions. We found that expression of Y397F-FAK/YCam in human astrocytoma cells decreases the level of phosphorylation of FAK at endogenous Tyr-397 residues and at both endogenous and exogenous Tyr-576 residues, in the putative activation loop of the kinase. This corresponds to a decrease in phosphorylation of FAK at focal adhesions in Y397F-FAK/YCam cells, since the cellular localisation of FAK phosphoTyr-576 in cells expressing Y397F-FAK/YCam or FAK/YCam was not different. Furthermore, FRAP analysis showed that phosphorylation of FAK at Tyr-397 increases specifically the time-residency of FAK at focal adhesions but not in cytosol. This in turn induces disassembly of focal adhesions at the cell tail and promotes cell motility as shown by the decrease in microtubule-mediated turnover of Y397F-FAK/YCam-containing focal adhesions. Our data show that phosphorylation of FAK at Tyr-397 is a key determinant of how FAK controls focal adhesion turnover.

Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions

Focal adhesion kinase (FAK) activity and Ca2+ signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca2+ sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca2+ variations at FA sites and FA dynamics. Discrete subcellular Ca2+ oscillators initiate both propagating and abortive Ca2+ waves in migrating U87 astrocytoma cells. Ca2+-dependent FA disassembly occurs when the Ca2+ wave reaches individual FAs, indicating that local but not global Ca2+ increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca2+ elevation. FAK-related non-kinase domain-Ycam had a faster, Ca2+-insensitive recovery half-time (11 s), which is consistent with the effect of Ca2+ on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca2+ and FAK autophosphorylation was correlated to intracellular Ca2+ levels, we propose that local Ca2+ elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion

FAK plays a key role in the regulation of cell migration. The authors show that the phosphorylation status of FAK at Tyr-925 is involved in FA turnover, formation of FAs, and increase in cell edge protrusion, together with activation of the p130CAS/Rac1 signaling pathway.

A Fluorescence Cell Biology Approach to Map the Second Integrin-binding Site of Talin to a 130-Amino Acid Sequence within the Rod Domain

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin β subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing αIIbβ3, linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984–2344), devoid of any known actin- or vinculin-binding sites, colocalized with β3-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin αIIbβ3 or with the recombinant wild type, but not the Y747A mutant β3 cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between β3-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984–2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for β3-cytoskeletal interactions but does not participate in αIIbβ3 activation.

The Small α5β1 Integrin Antagonist, SJ749, Reduces Proliferation and Clonogenicity of Human Astrocytoma Cells

The potential role of α 5 β 1 integrins in cancer has recently attracted much interest. However, few α 5 β 1 -selective antagonists have been developed compared with other integrins. The most specific nonpeptidic α 5 β 1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of α 5 β 1 . SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G 0 -G 1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more α 5 β 1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing α 5 . Treatment of α 5 -A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of α 5 β 1 expression. Our study highlights the importance of α 5 β 1 as an anticancer target and shows for the first time that a small nonpeptidic α 5 β 1 -specific antagonist affects proliferation of tumor cells. (Cancer Res 2006; 66(12): 6002-7)

Mechanism of calcium oscillations in migrating human astrocytoma cells.

Numerous studies show that intracellular calcium controls the migration rate of different mobile cell types. We studied migrating astrocytoma cells from two human cell lines, U-87MG and A172, in order to clarify the mechanisms by which calcium potentially influences cell migration. Using the wound-healing model to assay migration, we showed that four distinct components of migration could be distinguished: (i) a Ca(2+)/serum-dependent process; (ii) a Ca(2+)-dependent/serum-independent process; (iii) a Ca(2+)/serum-independent process; (iv) a Ca(2+)-independent/serum-dependent process. In U-87MG cells which lack a Ca(2+)-dependent/serum-independent component, we found that intracellular Ca(2+) oscillations are involved in Ca(2+)-dependent migration. Removing extracellular Ca(2+) greatly decreased the frequency of migration-associated Ca(2+) oscillations. Furthermore, non-selective inhibition of Ca(2+) channels by heavy metals such as Cd(2+) or La(3+) almost completely abolished changes in intracellular Ca(2+) observed during migration, indicating an essential role for Ca(2+) channels in the generation of these Ca(2+) oscillations. However, specific blockers of voltage-gated Ca(2+) channels, including nitrendipine, omega-conotoxin GVIA, omega-conotoxin MVIIC or low concentrations of Ni(2+) were without effect on Ca(2+) oscillations. We examined the role of internal Ca(2+) stores, showing that thapsigargin-sensitive Ca(2+) stores and InsP(3) receptors are involved in Ca(2+) oscillations, unlike ryanodine-sensitive Ca(2+) stores. Detailed analysis of the spatio-temporal aspect of the Ca(2+) oscillations revealed the existence of Ca(2+) waves initiated at the leading cell edge which propagate throughout the cell. Previously, we have shown that the frequency of Ca(2+) oscillations was reduced in the presence of inhibitory antibodies directed against beta3 integrin subunits. A simple model of a Ca(2+) oscillator is proposed, which may explain how the generation of Ca(2+) oscillations is linked to cell migration.

IRAS is an anti-apoptotic protein.

Abstract: Active cell death, also known as apoptosis, has been implicated in the pathophysiology of diseases such as cancer, heart failure and neurodegenerative disorders. We report the anti‐apoptotic function of IRAS, which was previously shown to bind imidazoline ligands. The amino acid sequence of human IRAS (hIRAS) is unrelated to known proteins, except for rat IRAS and a mouse homologue named nischarin, which binds the alpha5 integrin subunit of the fibronectin receptor. When stably transfected into PC12 cells, hIRAS localizes to the cytosol as a 167 kDa immunoreactive protein. Clonal PC12 cell lines expressing hIRAS displayed normal serum growth responses. However, hIRAS expression led to prolonged cell survival against known apoptotic stimuli: serum starvation or thapsigargin or staurosporine treatments. The apoptotic population of hIRAS‐expressing cells was significantly reduced, and this protection was achieved by a decrease in caspase‐3 activity, phosphatidylserine translocation, and nuclear fragmentation. Similar protective effect was obtained in COS7 cells transiently transfected with hIRAS. A partial activation of the PI3 kinase pathway is possibly implicated in the anti‐apoptotic effect of IRAS. Thus, IRAS appears to represent a previously unknown anti‐apoptotic protein involved in the regulation of cell survival.

FAK competes for Src to promote migration against invasion in melanoma cells.

Melanoma is one of the most deadly cancers because of its high propensity to metastasis, a process that requires migration and invasion of tumor cells driven by the regulated formation of adhesives structures like focal adhesions (FAs) and invasive structures like invadopodia. FAK, the major kinase of FAs, has been implicated in many cellular processes, including migration and invasion. In this study, we investigated the role of FAK in the regulation of invasion. We report that suppression of FAK in B16F10 melanoma cells led to increased invadopodia formation and invasion through Matrigel, but impaired migration. These effects are rescued by FAK WT but not by FAKY397F reexpression. Invadopodia formation requires local Src activation downstream of FAK and in a FAK phosphorylation-dependant manner. FAK deletion correlates with increased phosphorylation of Tks-5 (tyrosine kinase substrate with five SH3 domain) and reactive oxygen species production. In conclusion, our data show that FAK is able to mediate opposite effects on cell migration and invasion. Accordingly, beneficial effects of FAK inhibition are context dependent and may depend on the cell response to environmental cues and/or on the primary or secondary changes that melanoma experienced through the invasion cycle.

Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles

Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs). Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK) and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.

Targeting Focal Adhesion Kinase Using Inhibitors of Protein-Protein Interactions

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor protein tyrosine kinase that is overexpressed and activated in many human cancers. FAK transmits signals to a wide range of targets through both kinase-dependant and independent mechanism thereby playing essential roles in cell survival, proliferation, migration and invasion. In the past years, small molecules that inhibit FAK kinase function have been developed and show reduced cancer progression and metastasis in several preclinical models. Clinical trials have been conducted and these molecules display limited adverse effect in patients. FAK contain multiple functional domains and thus exhibit both important scaffolding functions. In this review, we describe the major FAK interactions relevant in cancer signalling and discuss how such knowledge provide rational for the development of Protein-Protein Interactions (PPI) inhibitors.