Notch signaling is controlled by ligand binding, which unfolds a negative control region to induce proteolytic cleavage of the receptor. First, a membrane-proximal cleavage is executed by a metalloprotease, removing the extracellular domain. This allows ␥-secretase to execute a second cleavage within the Notch transmembrane domain, which releases the intracellular domain to enter the nucleus. Here we show that the ADAM10 metalloprotease Kuzbanian, but not ADAM17/tumor necrosis factor ␣-converting enzyme, plays an essential role in executing ligand-induced extracellular cleavage at site 2 (S2) in cells and localizes this step to the plasma membrane. Importantly, genetic or pharmacological inhibition of metalloproteases still allowed extracellular cleavage of Notch, indicating the presence of unknown proteases with the ability to cleave at S2. Gain of function mutations identified in human cancers and in model organisms that map to the negative control region alleviate the requirement for ligand binding for extracellular cleavage to occur. Because cancer-causing Notch1 mutations also depend on (rate-limiting) S2 proteolysis, the identity of these alternative proteases has important implications for understanding Notch activation in normal and cancer cells.

Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little is known about binding partners. To identify Cx43-interacting proteins, we performed pull-down experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding to Cx43 is specific in that it is not observed with three other connexins. We established that a 35-amino acid juxtamembrane region in the Cx43 tail, which contains a presumptive tubulin binding motif, is necessary and sufficient for microtubule binding. Immunofluorescence and immunoelectron microscopy studies reveal that microtubules extend to Cx43-based gap junctions in contacted cells. However, intact microtubules are dispensable for the regulation of Cx43 gap-junctional communication. Our findings suggest that, in addition to its well-established role as a channel-forming protein, Cx43 can anchor microtubule distal ends to gap junctions and thereby might influence the properties of microtubules in contacted cells.

Phosphatidic acid (PA), an intriguing phospholipid that is rapidly produced during receptor-stimulated breakdown of phosphoinositides, has often been proposed to function as a Ca2+ ionophore in activated cells. The PA-ionophore hypothesis is supported by the fact that exogenously applied PA stimulates Ca2+ uptake in various cells and can evoke Ca2+-mediated physiological responses, but it is not known whether PA accumulation affects cytoplasmic free Ca2+ concentration ([Ca2+]i). Here we report that PA elicits a transient rise in [Ca2+]i in cultured cells, not by stimulating Ca2+ influx, but, surprisingly, by releasing Ca2+ from intracellular stores. We further show that PA evokes growth factor-like effects in that it raises cytoplasmic pH, induces expression of the c-fos and c-myc proto-oncogenes and stimulates DNA synthesis. Our results indicate that, unlike an ionophore, PA acts by triggering the hydrolysis of phosphoinositides, with consequent formation of second messengers such as inositol trisphosphate signalling Cai2+ release. Furthermore, our data strengthen the notion that any Ca2+-mobilizing stimulus acting through phospholipase C may ultimately function as a growth factor.