Unique little molecule inhibitors against the protein kinase activities of wildtype and mutant p110a.Protein Kinase IC50 (nM) p110 H1047R PIK-75 LY294002 A66 Wortmannin 99 2584 47 4150 E545K 101 ND ND 2320 p110a 56 ND ND ND p85 H1047R 67 2267 69 1960 E545K 18 1903 64 2490 p110a 27 757 47 2220 bic H1047R 15 6111 185 3240 E545K 84 2413 106 3480 p110a 52 1151 135ND = no signal detected. doi:10.1371/journal.pone.0071337.tPLOS 1 | plosone.orgOncogenic PI3K Has Greater Protein Kinase ActivityTable 2. The IC50 of unique smaller molecule inhibitors against the lipid kinase activity of wildtype and mutant p110a.Lipid Kinase IC50 (nM) H1047R PIK-75 LY294002 A66 Wortmannin 6 1836 41 3 E545K 24 528 30 three p110a 7 1986 34doi:10.1371/journal.pone.0071337.tdetailed explanation for the observed variations will demand in depth structural studies to define how protein substrates interact together with the kinase domain. In summary, we compared the in vitro protein kinase activity of all human class-1 PI 3-kinase isoforms as well as 2 frequent gainof-function oncogenic mutants of p110a, H1047R and E545K. We identified that the oncogenic mutants have stronger protein kinase activity as measured by autophosphorylation, as well as phosphorylation of the exogenous substrate, bic. Furthermore we show that the oncogenic mutants (in particular H1047R) are not as reliant on Mn2+ and retain significantly additional protein kinase activity in the presence of Mg2+ alone. We demonstrate that the protein kinaseactivity in the wildtype and oncogenic types of p110a are all inhibited by compact molecules known to inhibit the lipid kinase activity of p110a; which includes LY294002, A66 and to a lesser degree wortmannin. Nonetheless, inhibition was especially pronounced with PIK-75. Furthermore we showed that p110b and p110c exogenous and auto-phosphorylation was retained in the presence of Mg2+, and these isoforms have been relatively resistant to inhibition by all inhibitors except PIK-75 (and AS252424 for p110c). In contrast p110d was Mn2+ dependent, only weakly phosphorylated bic and was totally suppressed by all inhibitors utilized. Because PI 3-K phosphorylates exogenous substrates and has been linked to alterations in lipid kinase activity and probable activation of alternate signaling pathways [45], this elevated protein kinase activity of E545K and especially H1047R inside the presence of Mg2+ could have implications for the physiological activity of PI 3-K, specifically in tumours carrying these mutations.AcknowledgmentsWe thank Associate Professor Gordon Rewcastle for assistance with inhibitor production.Buy2820537-05-9 Author ContributionsConceived and designed the experiments: CB WJ JD MG PS.3,5-Dibromo-1H-pyrazole-4-carbonitrile web Performed the experiments: CB WJ JD JK.PMID:33716140 Analyzed the data: CB WJ PS. Contributed reagents/materials/analysis tools: JD JK MG PS. Wrote the paper: CB WJ JD MG JK PS.
Int. J. Mol. Sci. 2014, 15, 18437-18452; doi:ten.3390/ijmsOPEN ACCESSInternational Journal ofMolecular SciencesISSN 1422-0067 mdpi/journal/ijms ArticleMelatonin Attenuates Intermittent Hypoxia-Induced Lipid Peroxidation and Local Inflammation in Rat Adrenal MedullaYu Liu 1,two, George Lim Tipoe 3 and Man Lung Fung 1,*2Department of Physiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China; E-Mail: hannahliu8383@gmail Faculty of Medicine, Shenzhen University, Shenzhen 518060, Guangdong, China Division of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China; E-Mail: [email protected]* Author to whom.