Kenneth Takeda

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)

CD47 update: a multifaceted actor in the tumour microenvironment of potential therapeutic interest.

CD47 is a ubiquitous 50 kDa five‐spanning membrane receptor that belongs to the immunoglobulin superfamily. This receptor, also known as integrin‐associated protein, mediates cell‐to‐cell communication by ligation to transmembrane signal‐regulatory proteins SIRPα and SIRPγ and interacts with integrins. CD47 is also implicated in cell‐extracellular matrix interactions via ligation with thrombospondins. Furthermore, CD47 is involved in many and diverse cellular processes, including apoptosis, proliferation, adhesion and migration. It also plays a key role in many immune and cardiovascular responses. Thus, this multifaceted receptor might be a central actor in the tumour microenvironment. Solid tumours are composed of not only cancer cells that actively proliferate but also other cell types including immune cells and fibroblasts that make up the tumour microenvironment. Tumour cell proliferation is strongly sustained by continuous sprouting of new vessels, which also represents a gate for metastasis. Moreover, infiltration of inflammatory cells is observed in most neoplasms. Much evidence has accumulated indicating that infiltrating leukocytes promote cancer progression. Given its ubiquitous expression on all the different cell types that compose the tumour microenvironment, targeting CD47 could represent an original therapeutic strategy in the field of oncology. We present a current overview of the biological effects associated with CD47 on cancer cells and stromal cells.

Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca2+ responses in endothelial cells

Background and purpose: We previously reported that agonist‐induced rises in cytoplasmic Ca2+ concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short‐term (2 min) pre‐treatment with cAMP‐elevating agents. The aim of this work was to study the effects of longer term (8 h) pre‐treatment with dibutyryl‐cAMP (db‐cAMP) or rolipram, a specific inhibitor of phosphodiesterase‐4 (PDE4), on [Ca2+]i, cAMP levels and PDE activity and expression in HUVEC.

Voltage‐dependent currents and modulation of calcium channel expression in zona fasciculata cells from rat adrenal gland.

1. Whole‐cell voltage‐activated currents from single zona fasciculata (ZF) cells from rat adrenal glands were studied. T‐ and L‐type Ca2+ currents and a slowly inactivating A‐type K+ current were the three major currents observed. 2. In freshly isolated cells, the A‐type K+ current and the T‐type Ca2+ current were predominant. The A‐type current was activated at ‐50 mV and inhibited by 4‐amino‐pyridine with a half‐maximal block (IC50) at 130 microM while the T‐type current was activated at ‐70 mV and blocked by Cd2+, Ni2+ and amiloride with IC50 values of 24.1, 132.4 and 518.9 microM, respectively. 3. Under current clamp, depolarizing current pulses produced a single Ca2+ action potential with Cs+ in the pipette internal solution. Upon replacement of Cs+ by K+, the half‐amplitude width of the action potential was shortened and membrane potential oscillations were seen after the spike. 4. In freshly isolated cells and during the first 24 h after plating, the T‐type current was observed in all cells, with L‐type current being observed in < 2% of cells, even in the presence of (+)SDZ 202,791, a dihydropyridine Ca2+ channel agonist. With time in culture, the T‐type current disappeared, and a high‐voltage‐activated L‐type current became increasingly apparent. In cells tested after > 2 days in culture, (+)SDZ 202,791 potentiated L‐type current by 407 +/‐ 12% and the antagonist (‐)SDZ 202,791 blocked this increase. The L‐type current was activated between ‐30 and ‐20 mV and was sensitive to nitrendipine and omega‐conotoxin GVIA. 5. Pre‐incubation of cultured ZF cells with adrenocorticotrophic hormone (ACTH) or vasoactive intestinal peptide (VIP) for 3 days resulted in a high, sustained level of expression of T‐type current, with a mean amplitude of 34.2 +/‐ 5.5 pA pF‐1 for ACTH‐treated cells compared with 3.4 +/‐ 1.8 pA pF‐1 for untreated cells. Cycloheximide strongly inhibited this effect. Neither treatment affected L‐type current expression. 6. The expression of both Ca2+ current types was unaffected by pre‐incubation with 8‐bromo‐cAMP or forskolin. The protein kinase A antagonist, H89, did not inhibit the ACTH‐induced upregulation of T‐type Ca2+ currents. 7. It is concluded that the main voltage‐dependent currents involved in cell excitability and steroidogenesis in rat adrenal ZF cells are an A‐type K+ current and a T‐type Ca2+ current. The physiological role and control of expression of L‐type Ca2+ channels in rat ZF cells remain less clear.

The blocking effects of ω-conotoxin on Ca current in bovine chromaffin cells

The effects of ω-conotoxin (ω-CgTX) on voltage-sensitive Ca currents (ICa) were studied in cultured bovine adrenal chromaffin cells. A maximal block of ICa of 40–50% was obtained with ω-CgTX in the μM range, and was independent of the holding potential. The onset of block was both concentration- and time-dependent. In bovine chromaffin cells, Ca channels, both sensitive and insensitive to ω-CgTX, appear to be present.

Advanced glycation end products (AGEs) activate mast cells

BACKGROUND AND PURPOSE Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long‐lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells.