PPP2R2A is one of the regulatory subunits of the PP2A phosphatase complexes, and previous studies showed that its upregulation promotes cancer cell survival and growth. In this research, we used the tandem affinity purification and the HPLC-Chip-ESI/MS/MS mass spectrometry to screen the PPP2R2A-binding proteins and the results indicated that the GFPT-1/-2 were the potential partners of PPP2R2A. We further validated the interaction between PPP2R2A and GFPT-1/-2 through GST Pull-down, co-immunoprecipitation and immunofluorescence assays. And we found that knockdown of PPP2R2A by lentivirus-mediated shRNA enhanced the phosphorylation of GFPT2, whereas the phosphorylation of GFPT1 had no significant change. GFPT2 is a rate-limiting enzyme in the hexosamine pathway. Our results showed that the knockdown of PPP2R2A promoted the total cellular O-GlcNAcylation in MDA-MB-231 breast cancer cells. These results suggest that PPP2R2A interacts with GFPT1/2, and leads to the phosphorylation of GFPT2, which can regulate the cellular O-GlcNAcylation.
PPP2R2A 是PP2A 磷酸酶的调控亚基之一，以往的研究报道显示，PPP2R2A 可促进肿瘤细胞生存和生长。本研究通过串联亲和纯化联合HPLC-Chip-ESI/MS/MS 筛选PPP2R2A 的相互作用蛋白质，分析结果显示，L-谷氨酰胺-D-果糖-6-磷酸转氨酶1 (Glutamine-fructose-6-phosphate transaminase 1，GFPT1) 和L-谷氨酰胺-D-果糖-6-磷酸转氨酶2 (Glutamine-fructose-6-phosphate transaminase 2，GFPT2) 是PPP2R2A 可能的结合蛋白。通过免疫荧光共定位、GST Pull-down 和免疫共沉淀等方法，进一步确认了PPP2R2A 和GFPT1 及GFPT2 的相互结合。通过shRNA 下调PPP2R2A 后，GFPT2 的磷酸化水平显著增加，但GFPT1 的磷酸化水平改变不明显。GFPT2 是O-GlcNAC 糖基化修饰通路中的一个限速酶，在乳腺癌细胞MDA-MB-231 中下调PPP2R2A 后，蛋白质O-GlcNAC 糖基化修饰水平增加。这些结果表明，PPP2R2A 可直接结合GFPT2，并导致其去磷酸化，进而影响细胞内O-GlcNAC 糖基化修饰。.
Keywords: GFPT1; GFPT2; O-GlcNAcylation; PPP2R2A.