A High‐throughput Assay Platform for Quantifying Nucleo‐cytoplasmic Phosphatase Activity

Kinases and phosphatases are key regulators of cellular behavior; their combined activity determines the output of intracellular signaling cascades. To better understand phosphatase regulation, our lab has developed a set of high‐throughput, substrate‐specific phosphatase activity assays that profile the dynamics of cytoplasmic and nuclear phosphatase activity independently. Protein phosphorylation by kinases has been viewed as an active process requiring precise control while protein dephosphorylation by phosphatases has been thought of as a constitutive and unregulated process. Studies have shown that kinase regulation and specificity is important in determining cellular behavior. Only recently have similar observations been made for phosphatases. We now appreciate that phosphatase activity can be regulated post‐translationally and that their phosphosite specificity and subcellular localization are key determinants of intracellular signaling regulation. Previously, our lab has developed a whole cell phosphatase assay to better understand phosphatase regulation and quantified the dynamics of dual specificity phosphatase (DUSP) activities on MAPK substrates. We expanded the existing assay to quantify the activity of subcellularly partitioned phosphatases as well as phosphatases with different specificities including protein tyrosine phosphatases and protein serine/threonine phosphatases. We purified six recombinant phosphosubstrates: ERK2(T202/Y204), p38(T180/Y182), JNK(T183/Y185), MAPKAPK2(T334), CREB(S133), and STAT1(Y701). In a 96‐well format, each well is coated with a single phosphosubstrate and incubated with cytoplasmic or nuclear extracts containing active phosphatases. During cellular lysis extracts are ATP‐depleted to preclude kinase activity. Phosphatase activity is quantified by immunodetection and ELISA readout of the remaining unreacted phosphoprotein. We developed a lysis method that results in the cytoplasmic and nuclear fractionation of active phosphatases from adherent cell cultures. We validated compartmentalization of known cytoplasmic and nuclear proteins by western blot and showed compartment‐specific phosphatase measurements with our assay platform ( Fig 1 representative data). The low measurement noise of this assay (CV<20%) renders it sensitive and able to reliably detect phophatase activity with less than 50,000 cell lysates. Thus, we can quantify phosphatase activity toward all 6 substrates from a single extract making our assay amenable to parallelization. We demonstrate the power of our assay by profiling the kinetics of phosphatase activity in response to viral infection and cytokine stimulation. Our results show unique activity profiles depending on subcellular localization and substrate specificity. In this study we developed an assay platform to quantify nuclear and cytoplasmic substrate specific phosphatase activities. These assays are compatible with any adherent cell system and provides researchers with a method to study substrate specific phosphatase activity in a parallelizable and high‐throughput manner. Support or Funding Information This work was supported by the UVA Biotechnology Training Program, NIAID, and the American Heart Association. 1 Nucleo‐Cytoplasmic Fractionation and Phosphatase AssayA. Lysis schematic B. MCF10A WB C & D pMK2 Cyto and Nuc PPase Assay