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Serine Phosphorylation (serine + phosphorylation)
Selected AbstractsCalyculin A,induced actin phosphorylation and depolymerization in renal epithelial cellsCYTOSKELETON, Issue 4 2003Luo Gu This study reports actin phosphorylation and coincident actin cytoskeleton alterations in renal epithelial cell line, LLC-PK1. Serine phosphorylation of actin was first observed in vitro after the cell lysate was incubated with phosphatase inhibitors and ATP. Both the phosphorylated actin and actin kinase activities were found in the cytoskeletal fraction. Actin phosphorylation was later detected in living LLC-PK1 cells after incubation with the phosphatase inhibitor calyculin A. Calyculin A,induced actin phosphorylation was associated with reorganization of the actin cytoskeleton, including net actin depolymerization, loss of cell-cell junction and stress fiber F-actin filaments, and redistribution of F-actin filaments in the periphery of the rounded cells. Actin phosphorylation was abolished by 3-h ATP depletion but not by the non-specific kinase inhibitor staurosporine. These results demonstrate that renal epithelial cells contain kinase/phosphatase activities and actin can be phosphorylated in LLC-PK1 cells. Actin phosphorylation may play an important role in regulating the organization of the actin cytoskeleton in renal epithelium. Cell Motil. Cytoskeleton 54:286,295, 2003. © 2003 Wiley-Liss, Inc. [source] Oncostatin M,induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: An implication for the pathogenesis of arthritisARTHRITIS & RHEUMATISM, Issue 5 2009Sang-Heng Kok Objective To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. Methods The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. Results Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA,protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. Conclusion These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis. [source] Acute exercise reverses TRB3 expression in the skeletal muscle and ameliorates whole body insulin sensitivity in diabetic miceACTA PHYSIOLOGICA, Issue 1 2010A. Matos Abstract Aim:, TRB3 became of major interest in diabetes research when it was shown to interact with and inhibit the activity of Akt. Conversely, physical exercise has been linked to improved glucose homeostasis. Thus, the current study was designed to investigate the effects of acute exercise on TRB3 expression and whole body insulin sensitivity in obese diabetic mice. Methods:, Male leptin-deficient (ob/ob) mice swam for two 3-h-long bouts, separated by a 45-min rest period. After the second bout of exercise, food was withdrawn 6 h before antibody analysis. Eight hours after the exercise protocol, the mice were submitted to an insulin tolerance test (ITT). Gastrocnemius muscle samples were evaluated for insulin receptor (IR) and IRS-1 tyrosine phosphorylation, Akt serine phosphorylation, TRB3/Akt association and membrane GLUT4 expression. Results:, Western blot analysis showed that TRB3 expression was reduced in the gastrocnemius of leptin-deficient (ob/ob) mice submitted to exercise when compared with respective ob/ob mice at rest. In parallel, there was an increase in the insulin-signalling pathway in skeletal muscle from leptin-deficient mice after exercise. Furthermore, the GLUT4 membrane expression was increased in the muscle after the exercise protocol. Finally, a single session of exercise improved the glucose disappearance (KITT) rate in ob/ob mice. Conclusion:, Our results demonstrate that acute exercise reverses TRB3 expression and insulin signalling restoration in muscle. Thus, these results provide new insights into the mechanism by which physical activity ameliorates whole body insulin sensitivity in type 2 diabetes. [source] Overexpression of BAD preferentially augments anoikisINTERNATIONAL JOURNAL OF CANCER, Issue 2 2003Masashi Idogawa Abstract BAD is a BH3-only protein, and its proapoptotic activity is negatively regulated by serine phosphorylation. Here, we show that overexpression of BAD preferentially augments anchorage loss,induced apoptosis (anoikis). Gene transfer,mediated BAD overexpression alone did not induce apoptosis in attached MDCK cells but strongly augmented apoptosis when cells were cultured in suspension. In contrast, overexpression of another BH3-only protein, BID, displayed much lower augmentation of anoikis, suggesting a preferential contribution of BAD to anoikis. During suspension culture, unphosphorylated BAD was gradually increased and targeted to the mitochondria. Cotransfection of BAD with constitutively active Akt cDNA strongly inhibited this change. In contrast, the increase of unphosphorylated BAD was not significantly inhibited by several phosphatase inhibitors or cotransfection with a dominant negative calcineurin cDNA, implying that the increase may be mainly due to a decrease of serine kinase activity, such as that of Akt. Similar results were observed in COS-7 cells, suggesting that BAD overexpression can increase sensitivity of anchorage-dependent cancer cells to anoikis. Thus, we propose that BAD can serve as a valuable gene therapeutic molecule to inhibit carcinoma progression. © 2003 Wiley-Liss, Inc. [source] The cofilin activity cycle in lamellipodia and invadopodiaJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2009Matthew Oser Abstract The actin severing protein cofilin is essential for directed cell migration and chemotaxis, in many cell types and is also important for tumor cell invasion during metastasis. Through its severing activity, cofilin increases the number of free barbed ends to initiate actin polymerization for actin-based protrusion in two distinct subcellular compartments in invasive tumor cells: lamellipodia and invadopodia. Cofilin severing activity is tightly regulated and multiple mechanisms are utilized to regulate cofilin activity. In this prospect, we have grouped the primary on/off regulation into two broad categories, both of which are important for inhibiting cofilin from binding to F-actin or G-actin: (1) Blocking cofilin activity by the binding of cofilin to either PI(4,5)P2 at lamellipodia, or cortactin at invadopodia. (2) Blocking cofilin's ability to bind to actin via serine phosphorylation. Although the literature suggests that these cofilin regulatory mechanisms may be cell-type dependent, we propose the existence of a common cofilin activity cycle in which both operate. In this common cycle, the mechanism used to initiate cofilin activity is determined by the starting point in the cycle in a given subcellular compartment. J. Cell. Biochem. 108: 1252,1262, 2009. © 2009 Wiley-Liss, Inc. [source] 1141636674 Differential serine and tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) in Jeg-3 choriocarcinoma cell linesAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2006J Roediger Background:, Signal Transducer and Activator of Transcription 3 (STAT3) is an intracellular signalling molecule, which is used by several cytokines, including leukemia inhibitory factor (LIF), epithelial growth factor (EGF), and interleukin-6 (IL-6). It induces a variety of gene transcripts and cell functions. In trophoblast cells and in tumor cells, its tyrosine phosphorylation is directly linked to their invasiveness. The regulation and function of STAT3 serine phosphorylation is still widely unclear. Material and Methods:, Jeg-3 choriocarcinoma cells were stimulated with different concentrations of EGF, IL-6 and LIF. STAT3 serine (727) and tyrosine (705) phosphorylation were analyzed 5,60 min after stimulation by SDS-PAGE electrophoresis followed by Western blotting. Results:, Jeg-3 cells display spontaneous STAT3 serine phosphorylation. 100 ng/mL EGF induces a time-dependent reduction starting 15 min after stimulation. Tyrosine phosphorylation does not occur spontaneously, but is strongly induced by EGF at all analyzed time points. LIF induces tyrosine phosphorylation, but affects serine phosphorylation only very slightly. IL-6 did not influence neither serine phosphorylation nor tyrosine phosphorylation. Discussion:, The EGF induced STAT3 tyrosine phosphorylation may be responsible for its invasion triggering capacities. The parallel reduction of serine phosphorylation may enhance this effect. LIF was formerly shown to enhance trophoblast invasion via STAT3 tyrosine phosphorylation. IL-6 displays very little effects on STAT3 and seems to use other pathways for signalling. [source] Rapid activation of Mac-1(CD11b/CD18) molecules on macrophages by a new chemotactic factor ,Gasserokine' produced by Lactobacillus gasseri JCM1131TANIMAL SCIENCE JOURNAL, Issue 5 2002Haruki KITAZAWA ABSTRACT The chemoattractant activity of a new chemotactic factor, ,Gasserokine' produced by Lactobacillus gasseri JCM1131T, has been proposed as a novel immunological function of probiotic lactic acid bacteria. The focus of the present study was to understand the mechanism of the chemotaxis induced by Gasserokine, using activation of an adhesion molecule, Mac-1 (CD11b/CD18) on macrophages. The macrophage chemotaxis to Gasserokine was abolished by preincubation of macrophages with the anti-Mac-1 mAb. Gasserokine induced rapid serine phosphorylation of CD18 molecules within 1 min of stimulation, but the effect was short-lived. Substantial tyrosine phosphorylation was observed in CD18-associated protein of macrophages stimulated by Gasserokine. The tyrosine phosphorylation was confirmed in macrophages stimulated with Gasserokine and also serine/threonine phosphorylation was detected on CD18 molecules by laser microscopy using a double immunostaining method. These results suggest that selective activation of intracellular signaling cascades, such as the mitogen-activated protein kinase pathway, are related to the macrophage chemotaxis induced by Gasserokine. [source] | |||||||||||||