Identification of PP1–Gadd34 substrates involved in the unfolded protein response using K-BIPS, a method for phosphatase substrate identification

Abstract

Phosphorylation is a key post-translational modification in cell signaling, which is regulated by the equilibrium activities of kinases and phosphatases. The biological significance of many phosphorylation events remains poorly characterized due to the scarcity of tools to discover phosphatases substrates. In prior work, we established kinase-catalyzed biotinylation where kinases accept the γ-modified ATP analog, ATP-biotin, to label phosphoproteins. Here, we developed a novel method to study substrates of phosphatases using kinase-catalyzed biotinylation termed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In a proof-of-concept experiment, K-BIPS was initially used to explore the substrates of phosphatases inhibited by okadaic acid. Many known phosphatase substrates were observed, confirming K-BIPS as a valid phosphatase substrate identification tool. Then, as a further application, K-BIPS was used to discover the substrates of the PP1-Gadd34 phosphatase complex in the context of unfolded protein response (UPR). In addition to the known substrate eIF2α, K-BIPS revealed several novel substrates, suggesting a more prominent role for the PP1-Gadd34 complex in UPR than previously appreciated. Overall, the two studies establish K-BIPS as a powerful tool to discover the cellular substrates of phosphatases.

Delineating the role of eIF2α in retinal degeneration

“…This made us wonder if something else could be occurring in the retinas of rd16 Gadd34 −/− mice. Although eIF2α does not have any known function outside of protein synthesis regulation, GADD34 has a multitude of proposed functions outside of eIF2α regulation 13–15,33 . One particular function of GADD34 is its well-documented role as a pro-apoptotic protein 14,16,34 .…”

“…However, an elevation in p-eIF2α levels in the retinas of rd16 Gadd34 −/− mice was associated with a delay in photoreceptor cell death even in the absence of a change in the levels of protein synthesis. There are no known functions of eIF2α outside of protein synthesis regulation, but there are many other functions of GADD34 13–16,33 . Therefore, it is likely that the delay in RD observed rd16 Gadd34 −/− is due to the loss of GADD34 rather than the change in p-eIF2α levels.…”

“…Another important player of PP1 is CreP (PP1R15B), which is responsible for maintaining basal levels of p-eIF2a 12 . Aside from regulating eIF2α phosphorylation, GADD34 has a multitude of other roles including promoting apoptosis 13–16 . Interestingly, a recent study suggested that PP1/GADD34 may act on a plethora of previously unknown targets 13 .…”

“…Aside from regulating eIF2α phosphorylation, GADD34 has a multitude of other roles including promoting apoptosis 13–16 . Interestingly, a recent study suggested that PP1/GADD34 may act on a plethora of previously unknown targets 13 .…”

l‐Lactate Dehydrogenase Identified as a Protein Tyrosine Phosphatase 1B Substrate by Using K‐BIPS

“…As an additional method for phosphatase substrate identification, we previously developed K‐BIPS (kinase‐catalyzed biotinylation to identify phosphatase substrates) [20] . K‐BIPS relies on the fact that cellular kinases accept γ‐modified ATP analogues as co‐substrates.…”