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

Abstract

Kinases and phosphatases are major players in a variety of cellular events, including cell signaling. Aberrant activity or mutations in kinases and phosphatases can lead to diseases such as cancer, diabetes, and Alzheimer's. Compared to kinases, phosphatases are understudied; this is partly a result of the limited methods for identifying substrates. As a solution, we developed a proteomics-based method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) that previously identified substrates of Ser/Thr phosphatases using small molecule inhibitors. Here, for the first time, K-BIPS was applied to identify substrates of a tyrosine phosphatase, protein tyrosine phosphatase 1B (PTP1B), under siRNA knockdown conditions. Eight possible substrates of PTP1B were discovered in HEK293 cells, including the known substrate pyruvate kinase. In addition, l-lactate dehydrogenase (LDHA) was validated as a novel PTP1B substrate. With the ability to use knockdown conditions with Ser/Thr or Tyr phosphatases, K-BIPS represents a general discovery tool to explore phosphatases biology by identifying unanticipated substrates.

Identification of PP1c-PPP1R12A Substrates Using Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates

“…Prior work used small molecule inhibitors and knockdown to inactivate phosphatases for K-BIPS, resulting in the identification of multiple substrates of PP1-Gadd34 and PTP1B. 24,29 To complement prior work, the goal of this study was to use K-BIPS to identify substrates of the PP1c-PPP1R12A phosphatase complex. Moreover, this study used an inducible knockdown system to inactivate phosphatase activity for K-BIPS, which complements prior studies using inhibitor inactivation or knockdown via transient transfection.…”

“…Because substrates remained unlabeled without active phosphatases (Figure B, bottom panel), subsequent enrichment of biotinylated proteins (Figure D) and analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and label-free quantitation (Figure E) revealed biotinylated phosphatase substrates. Prior work used small molecule inhibitors and knockdown to inactivate phosphatases for K-BIPS, resulting in the identification of multiple substrates of PP1-Gadd34 and PTP1B. , …”

“…Prior work used small molecule inhibitors and knockdown to inactivate phosphatases for K-BIPS, resulting in the identification of multiple substrates of PP1-Gadd34 and PTP1B. 24 , 29 …”

“…Moreover, this study used an inducible knockdown system to inactivate phosphatase activity for K-BIPS, which complements prior studies using inhibitor inactivation or knockdown via transient transfection. 24 , 29 Using K-BIPS with PPP1R12A inducible knockdown in L6 myoblast cells, 136 candidate substrates and 14 high confidence hits were identified. The hit list included proteins with known direct or indirect interactions with the PP1c-PPP1R12A complex, as well as functions in cellular processes associated with PP1c-PPP1R12A in muscle cells, including cytoskeletal organization, cell adhesion, and cell division.…”