[Show abstract][Hide abstract] ABSTRACT: Background & Aims: Protein phosphatase 1 (PP1) is a member of the phosphoprotein phosphatase (PPP) family which, together with protein phosphatase 2A and protein phosphatase 2B (PP2B), exhibits serine/threonine phosphatase activity. We previously reported that linear alkylbenzenesulfonates (LAS) noncompetitively inhibit the phosphatase activity of calcineurin (CN), also known as PP2B. Here we examined the inhibitory effect of LAS on PP1α activity and determined the mechanism of inhibition.Methods: The inhibitory effect of LAS against PP1α enzymatic activity was confirmed using p -nitrophenylphosphate as a substrate and was further examined by kinetic analysis.Results: C12-LAS to C14-LAS exhibited strong inhibitory effects for PP1α. The half maximal inhibitory concentration (IC50) value of C12-LAS was 13.2μM. Using a double-reciprocal plot, C12-LAS was determined to exhibit uncompetitive inhibitory effects on PP1α activity.Conclusions: LAS analogues containing twelve to fourteen carbons in the alkyl side chain showed strong inhibitory effects against both PP1α and CN. Although LAS shows noncompetitive inhibition for CN, our results demonstrate that it exhibits uncompetitive inhibition for PP1α.
The Kitakanto Medical Journal 01/2015; 65(1):53-59. DOI:10.2974/kmj.65.53
[Show abstract][Hide abstract] ABSTRACT: Calcineurin (CN) is a Ca(2+)/calmodulin (CaM) dependent serine/threonine protein phosphatase and plays important role in several cellular functions in both higher and lower eukaryotes. Here we report inhibition of CN by linear alkylbenzene sulfonate. The clue to the finding was obtained while identifying the inhibitory material leaching from acrylonitrile butadiene rubber used for packing. Using standard dodecylbenzene sulfonate (C12-LAS), we obtained strong inhibition of CN with a half maximal inhibitory concentration of 9.3 µM, whereas analogs such as p-octylbenzene sulfonate and SDS hardly or only slightly affected CN activity. Three alkaline phosphatases, derived from shrimp, bacteria, and calf-intestine, which exhibit similar enzymatic activities to CN, were not inhibited by C12-LAS at concentrations of up to 100 µM. Furthermore, C12-LAS did not inhibit Ca(2+)/CaM-dependent myosin light chain kinase activity when tested at concentrations of up to 36 µM. The results indicate that C12-LAS is a potent selective inhibitor of CN activity.
[Show abstract][Hide abstract] ABSTRACT: Mn²⁺ is a minor nutrient, but is essential for numerous enzymatic activities and thus, for many cellular functions. However, its physiological roles and toxicity remain to be elucidated. In this study, we assessed the pharmacological potential and toxicity of Mn²⁺ in the immune system by examining the effects of Mn²⁺ on interleukin-2 (IL-2) production by Jurkat T-cells. Mn²⁺ at 0.1-1 mM did not significantly induce IL-2 production, whereas phorbol 12-myristate 13-acetate (PMA) at 1 μM slightly induced IL-2 production. Interestingly, Mn²⁺ at 0.3-0.7 mM promoted PMA-induced IL-2 production in a dose-dependent manner. A reporter gene assay revealed that Mn²⁺ promoted the activity of AP-1 (activator protein-1, a complex of c-Fos and c-Jun) in the presence of PMA. Western blot analysis showed that Mn²⁺ promoted the activation of JNK2 (c-Jun N-terminal kinase 2) and p38 MAPK (mitogen-activated protein kinase), which are both activators of AP-1, and upregulated the production of c-Fos and c-Jun within 4h in the presence of PMA. These results suggest that Mn²⁺ promotes PMA-induced IL-2 production by inducing the production and activation of AP-1, at least in part.
[Show abstract][Hide abstract] ABSTRACT: Calcineurin (CN) is thought to play an important role in the immune system by regulating cytokine production, for example, interleukin-2 (IL-2) in T-lymphocytes. We have previously shown that physiological concentrations of Zn2+ inhibit CN activity in vitro [K. Takahashi, E. Akaishi, Y. Abe, R. Ishikawa, S. Tanaka, K. Hosaka, Y. Kubohara, Zinc inhibits calcineurin activity in vitro by competing with nickel, Biochem. Biophys. Res. Commun. 307 (2003) 64-68], in spite of the fact that Zn2+ is an essential element of the CN catalytic domain. In this study, in order to assess whether Zn2+ regulates (suppresses) CN activity in vivo and whether Zn2+ can be used as an anti-inflammatory and/or immunosuppressive drug, we examined the effects of Zn2+ on IL-2 production induced by the mitogen, concanavalin A (ConA), in Jurkat T-cells. Zn2+ at 0.2 mM suppressed ConA-induced IL-2 accumulation in the medium of an in vitro culture of Jurkat cells. Zn2+ at 0.03-0.3 mM dose-dependently suppressed ConA-induced IL-2 mRNA expression in Jurkat cells. Zn2+ also suppressed IL-2 mRNA expression induced by phorbol ester (PMA) and ionomycin. Furthermore, Zn2+ and the immunosuppressant FK506 showed an additive inhibitory effect on ConA-induced IL-2 mRNA expression. These results suggest that exogenously added Zn2+ may disturb (increase) the intracellular Zn2+ concentration and inhibit CN activity, thereby suppressing IL-2 production in Jurkat cells. The present study further indicates that Zn2+ may have therapeutic potential in the treatment of T-cell related inflammation and also that Zn2+ may be utilized as a supplemental drug with FK506.
Biochemical and Biophysical Research Communications 10/2005; 335(1):162-7. DOI:10.1016/j.bbrc.2005.07.059 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.
[Show abstract][Hide abstract] ABSTRACT: Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.
Biochemical and Biophysical Research Communications 08/2003; 307(1):64-8. DOI:10.1016/S0006-291X(03)01122-7 · 2.28 Impact Factor