Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domaincontaining proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity. INTRODUCTIONThe GTPase dynamin plays a critical role in clathrin-mediated endocytosis (CME; Hinshaw, 2000). Dynamin self-assembles into a collar-like structure around the necks of deeply invaginated clathrin-coated pits (CCPs) where it is believed to directly mediate membrane fission and vesicle release. Dynamin is also associated with newly formed coated pits (Damke et al., 1994;Evergren et al., 2004) where it may function to regulate early stages of coated pit maturation (Sever et al., 2000a;Song and Schmid, 2003;Song et al., 2004).Although only one isoform of dynamin exists in Drosophila and in Caenorhabditis elegans, mammals express at least three isoforms, which are ϳ70% identical to each other. Dynamin-1, the first identified protein in the dynamin family, is expressed exclusively in neuronal cells where it functions in synaptic vesicle recycling (Shpetner and Vallee, 1989;Nakata et al., 1991;Sontag et al., 1994). Dynamin-2 is ubiquitously expressed (Cook et al., 1994;Sontag et al., 1994) and localizes to endocytic CCPs where it functions, like dynamin-1, in endocytosis (Damke et al., 1994;Altschuler et al., 1998). However, dynamin-2 may also be involved in vesicle formation at the Golgi Kreitzer et al., 2000), in regulating actin dynamics (Schafer, 2004), in cell signaling (Kranenburg et al., 1999;Fish et al., 2000), and has recently been localized to the centriole (Thompson et al., 2004). Dynamin-3, which is most highly expressed in testis but also detectable in neurons (Gray et al., 2003) and in lung (Nakata et al., 1993), has been less well studied.Dynamins are atypical GTPases, distinguished by their large size, low affinity for GTP, and high intrinsic rates of GTP hydrolysis (Sever et al., 2000b;Song and Schmid, 2003). Moreover, dynamin can self-assemble in solution (Hinshaw and Schmid, 1995) or on liposome templates into rings and spiral-like structures (Stowell et al., 199...

FGF-2 exerts its pleiotropic effects on cell growth and differentiation by interacting with specific cell surface receptors. In addition, exogenously added FGF-2 is translocated from outside the cell to the nucleus during G1-S transition. In this study, we show that a single point mutation in FGF-2 (substitution of residue serine 117 by alanine) is sufficient to drastically reduce its mitogenic activity without affecting its differentiation properties. The FGF-2(S117A) mutant binds to and activates tyrosine kinase receptors and induces MAPK and p70S6K activation as strongly as the wild-type FGF-2. We demonstrate that this mutant enters NIH3T3 cells, is translocated to the nucleus, and is phosphorylated similar to the wild-type growth factor. This suggests that FGF-2 mitogenic activity may require, in addition to signaling through cell surface receptors and nuclear translocation, activation of nuclear targets. We have previously shown that, in vitro, FGF-2 directly stimulates the activity of the casein kinase 2 (CK2), a ubiquitous serine/threonine kinase involved in the control of cell proliferation. We report that, in vivo, FGF-2(WT) transiently interacts with CK2 and stimulates its activity in the nucleus during G1-S transition in NIH3T3 cells. In contrast, the FGF-2(S117A) mutant fails to interact with CK2. Thus, our results show that FGF-2 mitogenic and differentiation activities can be dissociated by a single point mutation and that CK2 may be a new nuclear effector involved in FGF-2 mitogenic activity.-Bailly, K., Soulet, F., Leroy, D., Amalric, F., Bouche, G. Uncoupling of cell proliferation and differentiation activities of basic fibroblast growth factor (FGF-2).

Key Points• Apelin plays a key role in maintaining hemostasis through the regulation of platelet function.• Treatment of platelets with apelin inhibits aggregation and thrombus formation.Apelin peptide and its receptor APJ are directly implicated in various physiological processes ranging from cardiovascular homeostasis to immune signaling. Here, we show that apelin is a key player in hemostasis with an ability to inhibit thrombin-and collagenmediated platelet activation. Mice lacking apelin displayed a shorter bleeding time and a prothrombotic profile. Their platelets exhibited increased adhesion and a reduced occlusion time in venules, and displayed a higher aggregation rate after their activation by thrombin compared with wild-type platelets. Consequently, human and mouse platelets express apelin and its receptor APJ. Apelin directly interferes with thrombin-mediated signaling pathways and platelet activation, secretion, and aggregation, but not with ADP and thromboxane A 2 -mediated pathways. IV apelin administration induced excessive bleeding and prevented thrombosis in mice. Taken together, these findings suggest that apelin and/or APJ agonists could potentially be useful adducts in antiplatelet therapies and may provide a promising perspective for patients who continue to display adverse thrombotic events with current antiplatelet therapies. (Blood. 2016;127(7):908-920)