Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P₂) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P₂. This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P₂-induced talin activation. Finally, sequestration of PI4,5P₂ by a specific pleckstrin homology domain confirms that PI4,5P₂ is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P₂ exposes the integrin-binding site on talin. We propose that PI4,5P₂-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P₂ to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

We have shown previously that ADP released upon platelet adhesion mediated by α_IIbβ₃integrin triggers accumulation of phosphatidylinositol 3′,4′-bisphosphate (PtdIns-3,4-P₂) (Gironcel, D., Racaud-Sultan, C., Payrastre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253–256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-kinase in platelet morphological changes following adhesion, human platelets were pretreated with specific phosphoinositide 3-kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns-3,4-P₂synthesis was totally inhibited (25 μm LY294002 or 100 nm wortmannin), platelets adhered to the fibrinogen matrix, extended pseudopodia, but did not spread. Moreover, addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P₂ and dipalmitoyl phosphatidylinositol 3′,4′,5′-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4′,5′-bisphosphate (PtdIns-4,5-P₂) was able to trigger full spreading of wortmannin-treated adherent platelets. Following ³²P labeling of intact platelets, the recovery of [³²P]PtdIns-4,5-P₂ in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P₂ could be a downstream messenger of this signaling pathway.