Rosa Guasch | PubMed Health

PubMed2005

RhoE function is regulated by ROCK I-mediated phosphorylation.

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.

Animals↗

PubMed2003

Chronic ethanol consumption enhances interleukin-1-mediated signal transduction in rat liver and in cultured hepatocytes.

BACKGROUND: Interleukin-1 (IL-1) is a central mediator of the inflammatory process. Increased serum levels of IL-1 have been reported in alcoholics with liver damage, but it remains unknown whether chronic ethanol intake, in the presence or absence of lipopolysaccharide (LPS), activates IL-1 release and signaling in the hepatocyte. METHODS: IL-1beta and IL-10 release, expression of their receptors (IL-1RI and IL-10R), and the IL-1RI signal transduction response were evaluated in livers and cultured hepatocytes from ethanol-fed or pair-fed rats exposed in vivo or in vitro to LPS, ethanol, or both. RESULTS: Chronic ethanol intake increased both the serum levels of IL-1beta and IL-10 and the expression of IL-1RI, but not of IL-10R, in the liver microsomal fraction. In vivo LPS administration potentiated the ethanol-induced release of plasma cytokines. It is interesting to note that ethanol, either given in a single dose or chronically fed, stimulated IL-1beta and IL-10 release from cultured hepatocytes. Stimulation of hepatocytes with IL-1beta caused a higher activation of IL-1-associated kinase, extracellular receptor-activated kinases 1 and 2, and nuclear factor-kappaB (NF-kappaB) in hepatocytes from alcohol-fed animals than from controls. Furthermore, in the absence of any stimulation, hepatocytes from alcohol-fed animals showed an activation of both kinases, as well as an increase in NF-kappaB binding. Our results suggest the participation of the extracellular signal-regulated kinase (ERK)1/2 pathway in ethanol-induced NF-kappaB activation, because treatment with PD-98059, an ERK1/2 inhibitor, partially suppressed IL-1beta-induced NF-kappaB expression. CONCLUSIONS: Chronic ethanol intake potentiates the action of the proinflammatory cytokine IL-1beta, enhancing the release and signaling response of IL-1beta in the hepatocyte, which in conjunction with other cytokines or LPS may exacerbate the inflammatory damage associated with alcoholic liver disease.

Alcohol Drinking↗