Abstract
Protein Phosphatase 2A, PP2A, the principal Serine/threonine phosphatase, has major roles in broad range of signaling pathways that include regulation of cell cycle, cell proliferation and neuronal signaling. The loss of function of PP2A is linked with many human diseases, like cancer and neurodegenerative disorders. Protein phosphatase 2A (PP2A) functions as tumor suppressor and its tumor suppressor activity is inhibited by the overexpression of PP2A inhibitor proteins in most of the cancers. ARPP-19/ARPP-16 has been identified as one of the potential PP2A inhibitor proteins. Here, we report the resonance assignment of backbone 1H, 13C and 15N atoms of human ARPP-19 and ARPP-16 proteins. These chemical shift values can provide valuable information for the further study of the dynamics and interaction of ARPP-proteins to PP2A using NMR spectroscopy.
Highlights
ARPP-19 and ARPP-16 plays roles in the regulation of cell cycle
Recent studies have reported the role of ARPP-19 in the development and progression of several human cancer types, such as, breast cancer (Lü et al 2015), hepatocellular carcinoma (Song et al 2014) and human glioma (Jiang et al 2016)
ARPPs phosphorylated by the MAST3 (Microtubule Associated Ser/ Thr kinase 3) kinase, a homolog of MASTL/greatwall kinase (Gwl) kinase, selectively inhibits tumor suppressor phosphatase 2A (PP2A) holoenzyme containing B55α and B56δ (Andrade et al 2017)
Summary
The human cAMP regulated phosphoproteins, ARPP-16 and ARPP-19, were overexpressed in the Escherichia coli strain BL21 Gold from a plasmid vector carrying the gene conferring ampicillin resistance. The proteins were produced as Glutathione S-transferase (GST) fusion proteins. Escherichia coli BL21 Gold cells with ARPP-19/ARPP-16 plasmids were cultured in M9 minimal containing 100 μg/ml ampicillin at 37 °C, shaking the culture at 250 rpm until the OD at 600 nm was 0.6. The cells were cooled down to 25 °C and expression of GST fusion proteins were induced by adding 0.4 mM isopropyl β-d-1-thiogalactose at 25 °C for 20 h, shaking the culture at 250 rpm. GST was cleaved by Tobacco Etch Virus (TEV) protease (Invitrogen, Life Technologies) at 4 °C for 16 h and removed from the solution with the Glutathione Agarose. The proteins were concentrated with Amicon ultra centrifugal 3K filter device (Millipore)
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