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).