Phosphorylation of PBX2, a novel downstream target of mTORC1, is determined by GSK3 and PP1

Abstract

Mechanistic target of rapamycin complex 1 (mTORC1) is a serine-threonine kinase that is activated by extracellular signals, such as nutrients and growth factors. It plays a key role in the control of various biological processes, such as protein synthesis and energy metabolism by mediating or regulating the phosphorylation of multiple target molecules, some of which remain to be identified. We have here reanalysed a large-scale phosphoproteomics data set for mTORC1 target molecules and identified pre-B cell leukemia transcription factor 2 (PBX2) as such a novel target that is dephosphorylated downstream of mTORC1. We confirmed that PBX2, but not other members of the PBX family, is dephosphorylated in an mTORC1 activity-dependent manner. Furthermore, pharmacological and gene knockdown experiments revealed that glycogen synthase kinase 3 (GSK3) and protein phosphatase 1 (PP1) are responsible for the phosphorylation and dephosphorylation of PBX2, respectively. Our results thus suggest that the balance between the antagonistic actions of GSK3 and PP1 determines the phosphorylation status of PBX2 and its regulation by mTORC1.

Keywords: glycogen synthase kinase 3 (GSK3) Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; ERK, extracellular signal–regulated kinase; FOXK1, forkhead box K1; GSK3, glycogen synthase kinase 3; HA, hemagglutinin; LARP1, La-related protein 1; MEK, ERK kinase; mTORC1, mechanistic target of rapamycin complex 1; PBS, phosphate-buffered saline; PBX2, pre–B cell leukemia transcription factor 2; PI3K, phosphoinositide 3-kinase; PDK1, phosphoinositide-dependent protein kinase 1; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RAG, RAS-related GTP-binding protein; RHEB, Ras homolog enriched in Brain; shRNA, short hairpin RNA; siRNA, small interfering RNA; TBC1D7, TBC1 (TRE2-BUB2-CDC16) domain family member 7; TSC2, tuberous sclerosis complex 2; WT, wild-type; mechanistic target of rapamycin complex 1 (mTORC1); phosphorylation; pre–B cell leukemia transcription factor 2 (PBX2); protein phosphatase 1 (PP1).