Identification of PIM1 substrates reveals a role for NDRG1 phosphorylation in prostate cancer cellular migration and invasion

摘要

A Schematic of the peptide-capture technique used to identify analog-sensitive (AS) PIM1 substrates and phosphorylation sites in prostate cancer cells. AS PIM1 uses ATPγS, a bulky ATP analog, to thiophosphorylate substrates. Upper panel: thiophosphorylated substrates are alkylated by p-nitrobenzyl mesylate (PNBM) and recognized by an antibody to the thiophosphate ester moiety (ThioP). Lower panel: thiophosphorylated peptides are captured on a resin, eluted, and identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). B ATP analog optimization in LNCaP cells stably expressing either WT PIM1 or AS PIM1. Cells were treated with indicated ATP analog (ATP = ATPγS; PheET = N6-Phenylethyl ATPγS) in the presence of digitonin, a mild-permeabilizing agent. Alkylation was completed using PNBM. Whole-cell lysates were analyzed by western blot for the presence of thiophosphorylation (ThioP), PIM1 (PIM1), and HSP90 as loading control. C AS PIM1 thiophosphorylates endogenous AR in LNCaP cells. LNCaP-WT PIM1 and LNCaP-AS PIM1 cells were treated PhET-ATPγS as above. AR was immunoprecipitated and analyzed by western blot for the presence of thiophosphorylation (thioP) and AR. Input shows the protein levels in cells of PIM1, as well as the abundance of immunoprecipitated AR. Tubulin acts as a loading control. Western blots are representative of two independent experiments.