Activity‐based profiling of Phosphoprotein Phosphatases from Yeast to Humans
Authors: Kettenbach Arminja Nadine , Lyons Scott Parker , Jenkins Nicole Parker , Nasa Isha Parker 
Published: June 2021

A ‘tug‐of‐war’ between kinases and phosphatases establishes the phosphorylation states of proteins. Proper temporal and spatial coordination of these opposing activities is essential to maintain appropriate phosphorylation site occupancy and regulate cellular signaling. Deregulation of either kinase or phosphatase activities disrupts this balance and is commonly observed in human diseases, including cancer.

Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases. More than three‐quarters of all proteins are phosphorylated. The majority of serine and threonine dephosphorylation is carried out by the family of phosphoprotein phosphatases (PPPs). PPPs are among the most highly conserved proteins from yeast to human. In mammals, the PPP family consists of seven enzymes (PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7). PPPs are multimeric holoenzymes. PPP catalytic subunits interact with a diverse array of associated subunits (>200), endogenous inhibitors, and substrates. This combinatorial approach to PPP complexity generates specificity and allows for regulation. However, it also creates challenges for the study of PPP signaling and its responses to external cues and stresses, drug treatment, and pathological rewiring.

We have developed a quantitative chemical proteomic strategy for the systematic interrogation of endogenous PPPs. This strategy utilizes an immobilized non‐selective PPP inhibitor combined with mass spectrometry‐based proteomics for the efficient capture, identification, and quantification of endogenously expressed PPPs named Phosphatase Inhibitor Bead (PIB) capture by mass spectrometry (“PIB‐MS”)

We used this approach to investigate PPP signaling in breast and glioblastoma cell lines, mouse tissues, yeast species, and different phases of the cell cycle. We found that PPP expression distinguishes cell lines of different cancer types based on their origin. We identified new PPP subunits, and by using bioinformatics and experimental approaches, we linked them to catalytic subunits and validated their interaction.

Intriguingly, we found that inactive PPP complexes are not accessible for PIB capture. PPP activity is modulated by endogenous inhibitors that displace associated subunits, block the active site or alter substrate binding. Indeed, we found that non‐PIB bound PPP complexes were strongly reduced in phosphatase activity.

We extended this approach to PPP phosphorylation analysis and found that mitotic phosphorylation of the PP2A catalytic subunit by cyclin‐dependent kinase 1 (Cdk1) regulates PP2A holoenzyme composition and activity in mitosis.

In conclusion, we have developed a chemical proteomics strategy PIB‐MS for quantitative profiling of endogenous PPP catalytic and associated non‐catalytic subunits from cells and tissues of diverse eukaryotes in a single analysis. We have employed this strategy to identify new PPP subunits and substrates and a new regulatory mechanism governing PP2A activity in mitosis.

Support or Funding Information

This work was supported by grants from NIH/NIGMS (R35GM119455 and P20GM113132) and The V Foundation for Cancer Research (V2016‐022) to A.N.K.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.