srGAP2 deactivates RhoA to control the duration of thrombin-mediated endothelial permeability

The endothelial barrier is a tightly regulated gateway in the transport of material between circulation and the tissues. Inflammatory mediators such as thrombin are able to open paracellular spaces in the endothelial monolayer to allow the extravasation of plasma proteins and leukocytes. Here we show that the protein SLIT-ROBO Rho GTPase-activating protein 2 (srGAP2) plays a critical role in regulating the extent of thrombin-mediated opening. We show that srGAP2 is not required for normal barrier function in resting endothelial cells, but that depletion of srGAP2 significantly increases the magnitude and duration of junctional opening in response to thrombin. We show that srGAP2 acts to switch off RhoA signaling after the contraction phase of thrombin-induced permeability, allowing respreading of cells and reformation of the barrier. srGAP2 is also required for effective restoration of the barrier after treatment with two other vasoactive agents that active RhoA – TNFα and angiotensin II. Taken together, we show that srGAP2 has a general function in controlling RhoA signaling in endothelial permeability, acting to limit the degree and duration of opening, by triggering the switch from endothelial cell contraction to respreading

Cyclic-AMP Increases Nuclear Actin Monomer Which Promotes Proteasomal Degradation of RelA/p65 Leading to Anti-Inflammatory Effects

The second messenger, cAMP has potent immunosuppressive and anti-inflammatory actions. These have been attributed, in part, to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappa B (NF-κB). However, the mechanisms underlying the modulation of NF-κB activity by cAMP remain unclear. Here we demonstrate an important role for cAMP-mediated increase in nuclear actin monomer levels in inhibiting NF-κB activity. Elevated cAMP or forced expression of a nuclear localised polymerisation defective actin mutant (NLS-Actin(R62D)) inhibited basal and TNFα induced mRNA levels of NF-κB-dependent genes and NF-κB-dependent reporter gene activity. Elevated cAMP or NLS-Actin(R62D) did not affect NF-κB nuclear translocation but did reduce total cellular and nuclear RelA/p65 levels. Preventing the cAMP-induced increase in nuclear actin monomer, either by expressing a nuclear localised active mutant of the actin polymerising protein mDIA, silencing components of the nuclear actin import complex IPO9 and CFL1 or overexpressing the nuclear export complex XPO6, rescued RelA/p65 levels and NF-κB reporter gene activity in forskolin-stimulated cells. Elevated cAMP or NLS-Actin(R62D) reduced the half-life of RelA/p65, which was reversed by the proteasome inhibitor MG132. Accordingly, forskolin stimulated association of RelA/p65 with ubiquitin affinity beads, indicating increased ubiquitination of RelA/p65 or associated proteins. Taken together, our data demonstrate a novel mechanism underlying the anti-inflammatory effects of cAMP and highlight the important role played by nuclear actin in the regulation of inflammation

Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

Increased transient receptor potential canonical type 3 (TRPC3) channels have been observed in patients with essential hypertension. In the present study we tested the hypothesis that increased monocyte migration is associated with increased TRPC3 expression. Monocyte migration assay was performed in a microchemotaxis chamber using chemoattractants formylated peptide Met-Leu-Phe (fMLP) and tumor necrosis factor-α (TNF-α). Proteins were identified by immunoblotting and quantitative in-cell Western assay. The effects of TRP channel-inhibitor 2–aminoethoxydiphenylborane (2-APB) and small interfering RNA knockdown of TRPC3 were investigated. We observed an increased fMLP-induced migration of monocytes from hypertensive patients compared with normotensive control subjects (246±14% vs 151±10%). The TNF-α-induced migration of monocytes in patients with essential hypertension was also significantly increased compared to normotensive control subjects (221±20% vs 138±18%). In the presence of 2-APB or after siRNA knockdown of TRPC3 the fMLP-induced monocyte migration was significantly blocked. The fMLP-induced changes of cytosolic calcium were significantly increased in monocytes from hypertensive patients compared to normotensive control subjects. The fMLP-induced monocyte migration was significantly reduced in the presence of inhibitors of tyrosine kinase and phosphoinositide 3-kinase. We conclude that increased monocyte migration in patients with essential hypertension is associated with increased TRPC3 channels

Colliding Worlds: Asteroid research and the legitimization of war in space

Accepted versio

When it's apple blossom time in Normandy

(Published By J. H. Remick

Sun Dodgers. Vocal score

(Published By Francis, Day & Hunter

Site recognition and substrate screens for PKN family proteins

International audienceThe protein kinase C related kinases (PRKs also referred to as PKNs) are a kinase family important in diverse functions including migration and cytokinesis. Here, we have re-evaluated and compared specificity for PKN1 and PKN3, and assessed predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and 3 phosphorylate serine residues in sequence contexts that have an arginine at position -3. In contrast, PKN1 and 3 do not tolerate Arg in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP170; a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR, was identified. This identified threonine 654 in EGFR as the PKN1 phosphorylation site and this retains an arginine at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr-654 is shown to be modulated by PKN in vivo