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.

Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-Stransferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macromolecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Coimmunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16 INK4A gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16 INK4A. They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.

2؉increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. In the hierarchical organization of FA-associated proteins, FAK appears as an early component in FA formation (14). The integrin/adhesion-dependent increase in FAK Tyr 397 autophosphorylation relies on intermolecular FAK transphosphorylation (15) mediated by the formation of integrin clusters. The central role of FAK in the formation of a functional FA is also emphasized by its scaffolding function, allowing direct interaction and translocation of signaling proteins such as Src, growth factor receptor binding protein-2, paxillin, phospholipase C-␥, phosphatidylinositol 3-kinase, and p130cas (16) toward FAs. Furthermore, FAK interacts with FA structural proteins, including integrins, talin, ␣-actinin and tensin, and via paxillin with both vinculin and F-actin (17). In FAK-deficient cells, reduced motility is accompanied by an increased number of FAs (18), suggesting that FAK tyrosine kinase activity is involved in the regulation of FA turnover (19).

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.

Integrin-associated intracellular Ca2؉ oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. Cell migration is a cyclic process involving initial protrusion of the leading edge, formation of adhesive sites, contraction of the cell body, and release of adhesive sites at the cell rear (1). Adhesive sites are dynamic membrane structures that vary in size and composition during migration. Integrins, actin stress fibers (SFs) 1 and other structural proteins, and regulatory signaling molecules cluster at focal adhesions (2). Focal adhesions (FAs) serve as points of traction for contractile forces underlying forward cell movement and their dynamics are finely regulated. For example, FAs are highly motile in stationary fibroblasts but are largely stationary in migrating fibroblasts, thereby transducing contractile forces into movement (3). This suggests the existence of a molecular clutch that couples cytoskeleton-mediated traction and cell contraction. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNKFocal adhesion kinase (FAK) is activated and localized at FAs upon cell adhesion to the extracellular matrix (ECM; Refs. 4 and 5). Given the abundance of FAs and the reduced migration of fibroblasts from FAK null mice (6), FAK is likely involved in FA remodeling during migration. FAK-related nonKinase (FRNK), the non-catalytic C-terminal portion of FAK containing the FA targeting sequence, is also expressed as a separate dominant negative protein (7). The differential expression of FAK and FRNK is transcriptionally regulated, each of these proteins having distinct promoters within the FAK gene (8). Although the function of endogenous FRNK is not clear, FRNK has been used to alter signaling via endogenous FAK. When overexpressed in cells, FRNK acts as a negative regulator of FAK activity, inhibiting phosphorylation of FAK and different FAK-related processes, including cell cycle progression (9, 10), cell spreading on fibronectin (7, 11), and migration (12, 13). This suggests that the inhibitory effects of FRNK in migration might arise from altered FAK localization and phosphorylation.We and others reported that migration is dependent on Ca 2ϩ signaling in astrocytoma (14)

The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB.

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Abstract:The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca 2~influx. Whereas postsynaptic 5-HT 3 receptors induce depolarization, being permeant to Naãnd K~, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 n M 1-(m-chlorophenyl) biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca 2~concentration ([Ca2~]) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2~]ranged from 1.3 to 1 .6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT 3 receptor antagonist, but were insensitive to the removal of external free Na~(substituted with N-methyl-o-glucamine), to prior depolarization induced on addition of 20 mM K~, or to voltage-gated Ca 2~channel blockade by 10 p~M Co2~/Cd2õrbyl ,uMw-conotoxin MVIIC/1~tMw-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2ĩ nflux induced by 5-HT 3 receptor activation in NG1 08-15 cells by 1 ,tiM mCPBG was substantially reduced by 10.tMCo 2~/Cd2ãnd was completely blocked by 1 ,uM nitrendipine, an L-type Ca2~channel blocker. We conclude that in contrast to the perikaryal 5-HT 3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant. Key Words: Serotonin 5-HT3 receptorPresynaptic regulation-Synaptosomes-Confocal microscopy-Internal free Ca 2~concentration measurements-NG1O8-15 cells.