Marjolein J. Vliem

8-pCPT-2′-O-Me-cAMP-AM: An Improved Epac-Selective cAMP Analogue

Cyclic adenosine monophosphate (cAMP) is a common second messenger involved in the regulation of many different cellular processes through the activation of protein kinase A (PKA), exchange protein directly activated by cAMP (Epac) and cyclicnucleotide-regulated ion channels. Adenylyl cyclases are ACHTUNGTRENNUNGresponsible for catalysing the formation of cAMP from ATP. Levels of cAMP can be raised in cells in response to a large variety of extracellular stimuli, which act via receptors coupled to heterotrimeric G proteins, which stimulate the activity of adenylyl cyclase. In addition, cAMP levels are controlled by phosphodiesterases (PDE), which catalyse the degradation of cAMP to AMP. In cells, cAMP levels can be artificially elevated by forskolin, which activates adenylyl cyclase directly. Furthermore, cAMP levels can be raised by inhibiting PDEs. These approaches are commonly used in tissue culture experiments, but, by generating cAMP, they do not discriminate between the various target proteins that are activated. Alternatively, membrane-permeable cAMP analogues, which selectively interact with particular receptor proteins, can be applied. For example, signalling pathways activated by Epac and PKA can be ACHTUNGTRENNUNGdistinguished by using 8-pCPT-2’-O-Me-cAMP and 6-Bnz-cAMP, respectively. Epac is a guanine nucleotide exchange factor for the small G protein Rap. Rap cycles between a signalling-inactive GDPbound state and a signalling-active GTP-bound state. cAMP-activated Epac catalyses the exchange of Rap-bound GDP for GTP. Epac and Rap function in a number of different cellular processes including insulin secretion, inhibition of cell scattering, neurotransmitter release and cAMP-induced barrier function in endothelial cells. Even though 8-pCPT-2’-O-Me-cAMP has become a widely used tool in Epac-related research, its biological application is limited by its low membrane permeability, caused by the negatively charged phosphate. However, the negatively charged singly bonded oxygen on the phosphate group can be masked by labile esters. Such a precursor is expected to enter the cell efficiently, where the ester is hydrolysed either directly by water or by cellular esterases to liberate the active compound. We therefore synthesised 8-pCPT-2’-O-Me-cAMP-AM from 8pCPT-2’-O-Me-cAMP, whereby acetoxymethyl bromide was used as a donor for the AM group. The product that was obtained had a purity exceeding 97% and consisted of a mixture of the equatorial and the axial isomers of the ester (Figure S1 in the Supporting Information, Scheme 1). Even though the isomers could be resolved by repetitive analytical HPLC runs, efficient separation on a preparative scale was not possible. Orange peel acetylesterase and esterase from porcine liver cleaved the equatorial isomer about five times more efficiently than the axial isomer within minutes (data not shown). The pharmacokinetics of both isomers are thus expected to be similar, justifying the application of a mixture of both isomers to cells. In any case, the isomeric ratio of an individual synthesis can be easily quality controlled by P NMR (Figure S1). To compare the efficiency of 8-pCPT-2’-O-Me-cAMP-AM and 8-pCPT-2’-O-Me-cAMP in activating Epac1 in vivo, an Epac1based fluorescence resonance energy transfer (FRET) probe was used. In this assay, activation of Epac1 by the binding of cAMP to the Epac1-FRET probe is measured as a reduction in the FRET signal. A431 cells transfected with the FRET probe were stimulated with 8-pCPT-2’-O-Me-cAMP-AM or 8-pCPT-2’O-Me-cAMP (Figure 1). Stimulation of cells with 100 mm 8pCPT-2’-O-Me-cAMP resulted in a decrease of the FRET signal that was approximately one order of magnitude slower than the decrease obtained upon stimulation with 1 mm 8-pCPT-2’O-Me-cAMP-AM. Furthermore, activation of Epac1 following stimulation with 100 mm 8-pCPT-2’-O-Me-cAMP could be further enhanced by the addition of forskolin, whereas 1 mm 8pCPT-2’-O-Me-cAMP-AM induced maximal activity of Epac1 under the given conditions. The activation of Epac by 8-pCPT2’-O-Me-cAMP-AM occurs within one minute after application. This is comparable with the kinetics of forskolin-induced Epac activation, and thus 8-pCPT-2’-O-Me-cAMP-AM mimics the “natural” response time of the signalling pathway. The activity of endogenous Epac can be monitored by isolating selectively Rap·GTP from cell lysates. Primary human umbilical vein endothelial cells (HUVEC) were stimulated with different concentrations of 8-pCPT-2’-O-Me-cAMP and 8-pCPT-2’-OMe-cAMP-AM (Figure 2A). Partial activation of Rap was induced by 10 mm 8-pCPT-2’-O-Me-cAMP, and full activation of the G protein was stimulated by 100 mm 8-pCPT-2’-O-Me-cAMP. In contrast, treatment of the cells with just 0.1 mm 8-pCPT-2’-OMe-cAMP-AM was sufficient to induce full Rap activation. [a] M. J. Vliem, W.-J. Pannekoek, Dr. J. Riedl, M. R. H. Kooistra, Prof. Dr. J. L. Bos, Dr. H. Rehmann Department of Physiological Chemistry Centre for Biomedical Genetics and Cancer Genomics Centre University Medical Center Utrecht Universiteitsweg 100, 3584CG Utrecht (The Netherlands) Fax: (+31)88-75-68101 E-mail : j.l.bos@umcutrecht.nl h.rehmann@umcutrecht.nl [b] B. Ponsioen, Dr. K. Jalink Division of Cell Biology, The Netherlands Cancer Institute Amsterdam (The Netherlands) [c] Dr. F. Schwede, Dr. H.-G. Genieser BIOLOG Life Science Institute Flughafendamm 9a, 28071 Bremen (Germany) [] These authors contribute equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Rap2A links intestinal cell polarity to brush border formation

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P2 at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.

Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

ABSTRACT Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an additional mechanism of Epac1 recruitment, mediated by activated ezrin-radixin-moesin (ERM) proteins. Epac1 directly binds with its N-terminal 49 amino acids to ERM proteins in their open conformation. Receptor-induced activation of ERM proteins results in increased binding of Epac1 and consequently the clustered localization of Epac1 at the plasma membrane. Deletion of the N terminus of Epac1, as well as disruption of the Epac1-ERM interaction by an interfering radixin mutant or small interfering RNA (siRNA)-mediated depletion of the ERM proteins, impairs Epac1-mediated cell adhesion. We conclude that ERM proteins are involved in the spatial regulation of Epac1 and cooperate with cAMP- and Rap-mediated signaling to regulate adhesion to the extracellular matrix.

The RapGEF PDZ-GEF2 is required for maturation of cell–cell junctions

The small G-protein Rap1 is a critical regulator of cell-cell contacts and is activated by the remodeling of adherens junctions. Here we identify the Rap1 guanine nucleotide exchange factor PDZ-GEF2 as an upstream activator of Rap1 required for the maturation of adherens junctions in the lung carcinoma cells A549. Knockdown of PDZ-GEF2 results in the persistence of adhesion zippers at cell-cell contacts. Activation of Rap1A rescues junction maturation in absence of PDZ-GEF2, demonstrating that Rap1A is downstream of PDZ-GEF2 in this process. Moreover, depletion of Rap1A, but not Rap1B, impairs adherens junction maturation. siRNA for PDZ-GEF2 also lowers the levels of E-cadherin, an effect that can be mimicked by Rap1B, but not Rap1A siRNA. Since junctions in Rap1B depleted cells have a mature appearance, these data suggest that PDZ-GEF2 activates Rap1A and Rap1B to perform different functions. Our results present the first direct evidence that PDZ-GEF2 plays a critical role in the maturation of adherens junctions.

The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity.

Direct interaction between the catalytic domain of Epac1 and the nuclear pore component RanBP2 blocks Epac1 catalytic activity and downstream cAMP signaling.

Rap1 Spatially Controls ArhGAP29 To Inhibit Rho Signaling during Endothelial Barrier Regulation

ABSTRACT The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here, we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling that operates by successive protein translocations to and complex formation at the plasma membrane.

The small GTPase RALA controls JNK-mediated FOXO activation by regulation of a JIP1 scaffold complex

Background: Forkhead box O (FOXO) transcription factors affect life span and age-related diseases. Results: Evolutionarily conserved role for RALA in stress-induced assembly of a JIP1 scaffold complex to ensure FOXO activity. Conclusion: RALA regulates formation of a JIP1 scaffold complex to propagate JNK signaling toward FOXO4 in response to ROS. Significance: JIP1 is important for ROS-induced signaling from RALA to FOXO. FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

The small GTPase RALA controls c-Jun N-terminal kinase-mediated FOXO activation by regulation of a JIP1 scaffold complex.

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.

Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation.

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.

Multiple Rap1 effectors control Epac1-mediated tightening of endothelial junctions

ABSTRACT Epac1 and Rap1 mediate cAMP-induced tightening of endothelial junctions. We have previously found that one of the mechanisms is the inhibition of Rho-mediated tension in radial stress fibers by recruiting the RhoGAP ArhGAP29 in a complex containing the Rap1 effectors Rasip1 and Radil. However, other mechanisms have been proposed as well, most notably the induction of tension in circumferential actin cables by Cdc42 and its GEF FGD5. Here, we have investigated how Rap1 controls FGD5/Cdc42 and how this interconnects with Radil/Rasip1/ArhGAP29. Using endothelial barrier measurements, we show that Rho inhibition is not sufficient to explain the barrier stimulating effect of Rap1. Indeed, Cdc42-mediated tension is induced at cell-cell contacts upon Rap1 activation and this is required for endothelial barrier function. Depletion of potential Rap1 effectors identifies AF6 to mediate Rap1 enhanced tension and concomitant Rho-independent barrier function. When overexpressed in HEK293T cells, AF6 is found in a complex with FGD5 and Radil. From these results we conclude that Rap1 utilizes multiple pathways to control tightening of endothelial junctions, possibly through a multiprotein effector complex, in which AF6 functions to induce tension in circumferential actin cables.