60kDa Lysophospholipase, a New Sgk1 Molecular Partner Involved in the Regulation of ENaC

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling

SI113, a Specific Inhibitor of the Sgk1 Kinase Activity that Counteracts Cancer Cell Proliferation

Background/Aims: Published observations on serum and glucocorticoid regulated kinase 1 (Sgk1) knockout murine models and Sgk1-specific RNA silencing in the RKO human colon carcinoma cell line point to this kinase as a central player in colon carcinogenesis and in resistance to taxanes. Methods: By in vitro kinase activity inhibition assays, cell cycle and viability analysis in human cancer model systems, we describe the biologic effects of a recently identified kinase inhibitor, SI113, characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold, that shows specificity for Sgk1. Results: SI113 was able to inhibit in vitro cell growth in cancer cells derived from tumors with different origins. In RKO cells, this kinase inhibitor blocked insulin-dependent phosphorylation of the Sgk1 substrate Mdm2, the main regulator of p53 protein stability, and induced necrosis and apoptosis when used as a single agent. Finally, SI113 potentiated the effects of paclitaxel on cell viability. Conclusion: Since SI113 appears to be effective in inducing cell death in RKO cells, potentiating paclitaxel sensitivity, we believe that this new molecule could be efficiently employed, alone or in combination with paclitaxel, in colon cancer chemotherapy

The human asparaginase enzyme (ASPG) inhibits growth in leukemic cells

<div><p>The human protein ASPG is an enzyme with a putative antitumor activity. We generated in bacteria and then purified a recombinant GST-ASPG protein that we used to characterize the biochemical and cytotoxic properties of the human ASPG. We demonstrated that ASPG possesses asparaginase and PAF acetylhydrolase activities that depend on a critical threonine residue at position 19. Consistently, ASPG but not its T19A mutant showed cytotoxic activity in K562, NALM-6 and MOLT-4 leukemic cell lines but not in normal cells. Regarding the mechanism of action of ASPG, it was able to induce a significant apoptotic death in K562 cells. Taken together our data suggest that ASPG, combining different enzymatic activities, should be considered a promising anti-cancer agent for inhibiting the growth of leukemia cells.</p></div

Schematic representation of ASPG action on the growth and survival of leukemia cells.

<p>ASPG with its L-asparaginase activity could deprive leukemia cells of L-asparagine, which is an essential metabolite for its malignant growth, inducing apoptosis and blocking cell proliferation. ASPG could also inducing the arrest of cell proliferation converting the active PAF in the inactive form Lyso-PAF and down-regulating the expression of the Epithelial Sodium Channel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178174#pone.0178174.ref003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178174#pone.0178174.ref026" target="_blank">26</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178174#pone.0178174.ref027" target="_blank">27</a>].</p

Cytotoxicity of GST-ASPG on human leukemia cell lines and normal cells.

<p>The percentage of cell survival was evaluated by CCK8 assay in K562 (A), NALM-6 (B) and MOLT-4 (C) cell lines after 24h of treatment with GST-ASPG (●) and its inactive catalytically mutant GST-ASPG (T19A) (▲). HDFA (□) and PBMCs (◊) were used to analyze the cytotoxicity of GST-ASPG in normal cells (D). The results were fitted using GraphPad Prism software and represent the average and the standard deviation of three independent experiments. * (<i>p</i> < 0.05); *** (<i>p</i> < 0.001).</p

PAF-AH activity of GST-ASPG.

<p>(A) The PAF-AH activity of recombinant GST-ASPG (●) and its point mutant GST-ASPG (T19A) (▲) was measured as function of enzyme concentration using Abcam's PAF acetylhydrolase Assay Kit as reported in Materials and Methods. Data points are represented as means ± SD of triplicate sample measurements and were fitted with a second order polynomial equation in GraphPad Prism software. (B) The residual PAF-acetylhydrolase activity of GST-ASPG was measured after pre-incubation for 10 min of 1.5 μg of GST-ASPG with 40 mM and 200 mM of D-asparagine using Abcam's PAF-AH assay. Data are shown as means ± SD of triplicate measurements. ** (<i>p</i> < 0.01); *** (<i>p</i> < 0.001).</p

SDS Page of purified GST-fusion proteins.

<p>Line 1: marker, line 2: GST, line 3: GST-ASPG, line 4: GST-ASPG (T19A), line 5–7: 50, 100 and 250 ng of bovine serum albumin respectively.</p

Cell growth inhibition by GST-ASPG.

<p>(A) K562 cells after 24h of incubation with 1000 ng (115 nM) of GST-ASPG and its inactive mutant GST-ASPG (T19A). (B) Time course of K562 cells treatment with 115 nM of GST-ASPG (T19A) (▲), 115 nM of GST ASPG (●) and after further addition of 115 nM of GST-ASPG at 12h (ο).Viability of K562 (C) and NALM-6 (D) cells after 24h of treatment with increasing concentrations of GST-ASPG (●) and its inactive catalytically mutant GST-ASPG (T19A) (▲). The results (the average and the standard deviation of three independent experiments) were evaluated by Trypan Blue exclusion assay and fitted using GraphPad Prism software. * (<i>p</i> < 0.05); ** (<i>p</i> < 0.01); *** (<i>p</i> < 0.001).</p

Characterization of L-asparaginase activity of GST-ASPG.

<p>L-Asparaginase activity of GST-ASPG (●) and its catalytically inactive mutant GST-ASPG (T19A) (▲) was evaluated by Nessler’s method in a time dependent manner (A), as a function of enzyme concentration (B) and substrate concentration (C). A hill slope of 6.9 and an S_0.5 value of ≈13 mM were estimated. Ammonia release of GST-ASPG was measured using 15 mM of L-asparagine alone or in presence of 15 mM of D-asparagine; No detectable enzymatic activity was found using 15 mM of D-asparagine or 15 mM of L-glutamine as substrates (D). Steady state kinetic of recombinant GST-ASPG was calculated in presence of 0 mM (●), 10 mM (■) and 30 mM (▲) of D-asparagine and a Ki value of 71 mM was estimated (E). All the experiments were performed at 37°C as reported in Materials and Methods using 1.5 μg of each protein; data points are represented as means ± SD of triplicate sample measurements. *** (<i>p</i> < 0.001).</p