Browsing by Subject "ERK1/2"

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Identification and functional studies of two novel serine phosphorylation sites of insulin receptor substrate (IRS)-2: Ser 675 and Ser 907
(2010) Fritsche, Louise; Schleicher, Erwin D.
Insulin receptor substrate (IRS) proteins are major transducers of the insulin and IGF-1 signal into the PI-3 kinase/PKB and the MAP kinase pathway. In addition to tyrosine phosphoryla-tion, a large number of serine/threonine phosphorylation sites enable the IRS proteins to inte-grate different extra- and intracellular stimuli resulting in positive and negative modulation of the insulin and IGF-1 signal. Chronic hyperphosphorylation of serine/threonine sites of IRS-1 is involved in the development of insulin resistance. IRS-2 is of great importance for β-cell survival and for the regulation of hepatic metabolism. The study of serine/threonine phos-phorylations is required to understand the physiological and pathophysiological regulation of this important mediator of insulin signaling. In this thesis two novel IRS-2 serine phosphoryla-tion sites have been identified and characterized (mouse amino acid numbering): Ser 675, which is located in the kinase regulatory loop binding (KRLB) domain unique to IRS-2 and Ser 907, which is adjacent to the Grb2 binding site Tyr 911. Using phospho-site specific antibod-ies both sites were demonstrated to be phosphorylated upon insulin, phorbol ester and ani-somycin treatment in Fao rat hepatoma cells. The phosphorylation was also detected in pri-mary human hepatocytes and in liver tissue of insulin treated or refed mice. The insulin-induced phosphorylation of Ser 907 was mediated by the MAP kinase ERK1/2. Simulation of a permanent phosphorylation of this site in BHK cells expressing IRS-2 Glu 907 led to a slight decrease of IRS-2 tyrosine phosphorylation with no apparent effect on insulin downstream signaling. The insulin-induced association of IRS-2 with Grb2 in HEK293 cells was abrogated by mutation of the adjacent Tyr 911 to Phe, but not influenced by mutation of Ser 907 to Ala. Of note, the activation of MAP kinase signaling was not impaired in HEK293 cells expressing IRS-2 Phe 911 and not regulated by the expression level of IRS-2 wildtype, but completely dependent on IR expression, indicating the importance of an alternative, IRS-2-Grb2-independent pathway for the activation of MAP kinase signaling in these cells. The insulin-induced phosphorylation of Ser 675 was dependent on mTOR, but not on the downstream kinase p70 S6K1. Prevention of this phosphorylation in BHK cells or HEK293 cells expressing IRS-2 Ala 675 had no effect on proximal or distal insulin signal transduction. But compared with IRS-2 wildtype, the mutated IRS-2 protein Ala 675 showed increased half life in cycloheximide-treated HEK293 cells. Thus, phosphorylation of Ser 675 could have a similar function as its homologous site Ser 632 in IRS-1 and could be involved in the regula-tion of mTOR-dependent IRS-2 proteasom-mediated protein degradation.
Resveratrol is a natural inhibitor of human intestinal mast cell activation and phosphorylation of mitochondrial ERK1/2 and STAT3
(2021) Bilotta, Sabrina; Paruchuru, Lakshmi Bhargavi; Feilhauer, Katharina; Köninger, Jörg; Lorentz, Axel
Mast cells play a critical role as main effector cells in allergic and other inflammatory diseases. Usage of anti-inflammatory nutraceuticals could be of interest for affected patients. Resveratrol, a natural polyphenol found in red grapes, is known for its positive properties. Here, we analyzed the effects of resveratrol on FcεRI-mediated activation of mature human mast cells isolated from intestinal tissue (hiMC). Resveratrol inhibited degranulation and expression of cytokines and chemokines such as CXCL8, CCL2, CCL3, CCL4, and TNF-α in a dose-dependent manner. Further, resveratrol inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and signal transducer and activator of transcription (STAT) 3. ERK1/2 is known to be involved in cytokine expression of hiMC and to directly interact with STAT3. Mitochondrial STAT3 is phosphorylated by ERK1/2 and contributes to mast cell degranulation. We were able to isolate mitochondrial fractions from small hiMC numbers and could show that activation of mitochondrial STAT3 and ERK1/2 in hiMC was also inhibited by resveratrol. Our results indicate that resveratrol inhibits hiMC activation by inhibiting the phosphorylation of mitochondrial and nuclear ERK1/2 and STAT3, and it could be considered as an anti-inflammatory nutraceutical in the treatment of mast cell-associated diseases.