Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2-O-methyladenosine-3, 5-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.

Insulin receptor tyrosine kinase substrate p53 (IRSp53) has been identified as an SH3 domain-containing adaptor that links Rac1 with a Wiskott-Aldrich syndrome family verprolin-homologous protein 2 (WAVE2) to induce lamellipodia or Cdc42 with Mena to induce filopodia. The recruitment of these SH3-binding partners by IRSp53 is thought to be crucial for F-actin rearrangements. Here, we show that the N-terminal predicted helical stretch of 250 amino acids of IRSp53 is an evolutionarily conserved F-actin bundling domain involved in filopodium formation. Five proteins including IRSp53 and missing in metastasis (MIM) protein share this unique domain and are highly conserved in vertebrates. We named the conserved domain IRSp53/MIM homology domain (IMD). The IMD has domain relatives in invertebrates but does not show obvious homology to any known actin interacting proteins. The IMD alone, derived from either IRSp53 or MIM, induced filopodia in HeLa cells and the formation of tightly packed parallel F-actin bundles in vitro. These results suggest that IRSp53 and MIM belong to a novel actin bundling protein family. Furthermore, we found that filopodium-inducing IMD activity in the full-length IRSp53 was regulated by active Cdc42 and Rac1. The SH3 domain was not necessary for IMD-induced filopodium formation. Our results indicate that IRSp53, when activated by small GTPases, participates in F-actin reorganization not only in an SH3-dependent manner but also in a manner dependent on the activity of the IMD.Insulin receptor tyrosine kinase substrate p53 (IRSp53), 1 also known as brain-specific angiogenesis inhibitor 1-associated protein 2, is a multifunctional adaptor protein enriched in the central nervous system (1-3). The protein contains a unique N-terminal 250-amino acid stretch, a half-Cdc42/Rac interactive binding (CRIB) motif, a proline-rich domain, a Src homology 3 (SH3) domain, and a WW domain-binding motif (WWB). IRSp53 is directly regulated by Rho family small GTPases Rac1 and Cdc42 and provides a molecular link between these GTPases and the actin cytoskeleton regulators WiskottAldrich syndrome protein (WASP) family verprolin homologous protein 2 (WAVE2) and mammalian enabled (Mena), which are involved in the formation of lamellipodia (4, 5) and filopodia (6, 7). Active Cdc42 binds to the half-CRIB motif (6, 7), whereas Rac1 binds to the unique N-terminal domain (8). The association of Rac1 or Cdc42 is proposed to liberate the C-terminal SH3 domain masked intramolecularly by its N terminus, thereby allowing the SH3 domain to interact with its binding partners (4, 7, 9). Thus, the SH3 domain is thought to be essential for IRSp53-mediated actin reorganization. However, the N-terminal half of IRSp53 lacking the SH3 domain was reported to induce neurite outgrowth in a neuroblastoma cell line (6) and filopodia in B16 melanoma cells (10), suggesting that IRSp53 promotes actin reorganization independently of SH3 domain-mediated intermolecular interactions.Recently, a novel monomeric actin-binding protein, missing in met...

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherindependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with ␤-catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca 2؉ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca 2؉ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion. INTRODUCTIONIntercellular adhesion of vascular endothelial cells is essential for connecting neighboring endothelial cells to develop a vascular tree and to function as a barrier separating blood and tissues. Vascular endothelial cell adhesion is characterized by the overlapping of adherens junctions (AJs) and tight junctions (TJs). AJs are constituted by vascular endothelial cadherin (VE-cadherin) in close cooperation with platelet and endothelial adhesion molecule-1 (PECAM-1) and nectin. VE-cadherin-mediated cell adhesion depends on extracellular Ca 2ϩ , but not those mediated by PECAM-1 and nectin. TJs are made up of junctional adhesion molecule (JAM) family members, occuludin, claudin-5, and nectin (reviewed in Dejana, 2004).VE-cadherin has an extracellular domain constituted by five cadherin domains, a transmembrane domain, and a cytoplasmic domain connected to p120 catenin and ␤-catenin (Iyer et al., 2004). Through ␤-catenin, VE-cadherin is linked to ␣-catenin that is associated with the actin cytoskeleton, which results in the maintenance of cell-cell adhesion in conjunction with cytoskeleton (Herren et al., 1998;Navarro et al., 1998;Kobielak and Fuchs, 2004). Tyrosine-phosphorylated VE-cadherin in its cytoplasmic domain provides docking sites for signal-transmitting molecules (Esser et al., 1998;Zanetti et al., 2002;Hudry-Clergeon et al., 2005). Conversely, cytoplasmic domain modified by phosphorylation or associated with signaling molecules triggers the insideout signal that regulates the VE-cadherin-mediated cell adhesion (Nwariaku et al., 2004). ␤-catenin binds to other signaling molecules including PI3-K and MAGUK with inverted domain structure-1 (MAGI-1) as well as ␣-catenin (Kotelevets et al., 2005).MAGI-1 consists of six PSD95/DiscLarge/ZO-1 (PDZ) domains, a guanylate ...

Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130Cas , but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1-induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1-induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1-induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1-induced human aortic endothelial cells migration.

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.