The precise mechanisms of Ser-726 phosphorylation need to be determined in future studies. Acknowledgments We are grateful to Dr. of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes. (6) and (7) suggesting a relevance of adducin in proper assembly of F-actin bundles at intercellular junctions (8). The interaction of adducin with spectrin and actin is regulated by Ca2+ and calmodulin (4, 9) and through phosphorylation by various protein kinases such as protein kinase A (PKA), protein kinase C (PKC) (6, 10), and Rho-kinase (11, 12). It is noteworthy that Rho-GTPases are both important for actin cytoskeleton regulation (13,C15) and are involved in signaling induced by autoantibodies in pemphigus (16,C18). Pemphigus vulgaris (PV)3 is an autoimmune disease of the skin caused by autoantibodies directed against the adhesion molecules desmoglein (Dsg) 1 and 3 (19). There is growing evidence that both direct inhibition of Dsg3 interaction by antibody binding as well as intracellular signaling are necessary for Flurandrenolide intraepidermal blister formation (20). It has been shown that loss of keratinocyte cohesion in response to PV-IgG was accompanied by profound alterations of the cortical actin belt including fragmentation of actin filament bundles and increased stress fiber formation (21, 22). Pharmacological inhibition of p38 MAPK (21) or activation of RhoA (16,C18) was sufficient to block the PV-IgG-mediated loss of cell adhesion as well as effects on actin cytoskeleton reorganization. Moreover, for RhoA-mediated protection against autoantibody-induced loss of cell cohesion a requirement of cortical actin polymerization has been demonstrated (23). Concomitantly, a recent study reported a direct association of Dsg3 with actin and its involvement in actin dynamics (24). In the first part of the present study, we focused on the role of adducin for desmosomal keratinocyte cohesion Flurandrenolide and on the turnover of Dsg3. Because we identified adducin to become phosphorylated in response to pemphigus autoantibodies, we tested the contribution Flurandrenolide of several known PV-relevant signaling molecules for adducin phosphorylation in the second part of the study. EXPERIMENTAL PROCEDURES Cell Culture and Test Reagents The spontaneously immortalized human keratinocyte cell line HaCaT was grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), 50 units/ml of penicillin, and 50 g/ml of streptomycin (both AppliChem, Darmstadt, Germany), and maintained in a humidified atmosphere containing 5% CO2 at 37 C. For all experiments 1 105 cells/cm2 were seeded and grown in uncoated 24-well plates (Greiner Bio-One, Kremsmuenster, Austria) to confluence in high calcium medium (1.8 mm CaCl2x2H2O) within 4 days. The PV serum was drawn from a patient with active PV suffering from both oral and skin lesions. The ELISA values (Euroimmun, Luebeck, Germany) were 11,550 (Dsg3) and 375 (Dsg1) units/ml. Purification of IgG was performed as described previously (22), and IgG fractions were applied at 0.5 mg/ml. AK23, a monoclonal pathogenic antibody derived from a pemphigus mouse model (25), was purchased from Biozol, Eching, Germany, and used at 75 g/ml. A IgG fraction of a healthy volunteer was applied at 0.5 mg/ml. cytotoxic necrotizing factor (CNF)-1 (activation of RhoA, Rac1 and Cdc42) and CNFy (activation of RhoA) were purchased from Cytoskeleton (Denver, CO) and preincubated at a dose of 1 1 mm for 6 h. Rho-kinase was effectively blocked by application of y27632 (Merck, Darmstadt, Germany) at a dose of 30 m for 60 min. The p38 MAPK inhibitor SB202190 (Merck) was used at 30 m for 30 min or 24 h, respectively. The PKC activator Mouse monoclonal to RFP Tag phorbol 12-myristate 13-acetate (PMA) (Sigma) was applied at 50 nm, the inhibitor BIM-X (Enzo Life Sciences, Loerrach, Germany) at 1 m, and both were preincubated for 30 min. The PKA specific inhibitor H-89 (Sigma) was used at 10 m and preincubated for 2 h. BAPTA-AM (Sigma), a cell-permeable Ca2+ chelator, was applied at 50 m for 4 h. Transfection Adducin-specific small-interfering RNA (-adducin siRNA: L-009487-00-0005 or -adducin siRNA: L-008468-00-0005) was purchased from Thermo Fisher Scientific/Dharmacon, Layfatte, IN. The plasmids pEGFP-C1–adducin, pEGFP-C1–adducin (S726A), pEGFP-C1–adducin (S716A/S726A), and pEGFP-C1–adducin (S726D) were kindly provided by Hong-Chen Chen (National Chung Hsing University, Taichung, Taiwan) (26). The.