Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demonstrate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKC theta, a Ca(2+)-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3 tau isoform in vitro and in intact T cells. PKC theta and 14-3-3 tau coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3 tau interacted directly with purified PKC theta in vitro. Transient overexpression of 14-3-3 tau suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotransfected wild-type or constitutively active PKC theta, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca(2+)-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3 tau under similar conditions. Overexpression of wild-type 14-3-3 tau also inhibited phorbol ester-induced PKC theta translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3 tau caused increased localization of PKC theta in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3 tau was more effective than wild-type 14-3-3 tau in suppressing PKC theta-dependent IL-2 promoter activity, suggesting that 14-3-3 tau inhibits the function of PKC theta not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKC theta may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKC theta-mediated functions during T-cell activation.
ABSTRACT.The relationship between the p38-mitogen-activated protein kinase (p38-MAPK) signal pathway and high glucose-induced hepatic stellate cell (HSC) activation was investigated in this study. Sixty human HSC samples were randomly selected and used in the control (cultured normally), high-glucose (cultured in the presence of high glucose), and blocking (cultured under high-glucose conditions in the presence of the p38-MAPK inhibitor, SB203580) groups. The cells were incubated for 120 h and subsequently analyzed for morphological changes by inverted microscopy and for a-smooth muscle actin (a-SMA) expression (to determine the degree of HSC activation) by the method of streptavidin-biotin complex and western blot. Phospho-p38-MAPK protein expression was analyzed by western blotting. a-SMA and phospho-p38-MAPK expression was significantly upregulated in HSCs cultured under high-glucose conditions, compared to the HSCs cultured normally (P < 0.01). On the other hand, phospho-p38-MAPK and a-SMA protein levels were significantly lower in the blocking group compared to the high-glucose group (P < 0.01). Based on these results, we concluded that high-glucose levels induce HSC activation mediated by phospho-p38-MAPK. Therefore, blocking the p38-MAPK signal pathway could inhibit this effect.
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