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.
HDAC1 is the prototypical human histone deacetylase (HDAC) enzyme responsible for catalyzing the removal of acetyl group from lysine residues on many substrate proteins. By deacetylating histones and non-histone proteins, HDAC1 has a profound effect on the regulation of gene transcription and many processes related to cell growth and cell death, including cell cycle progression, DNA repair, and apoptosis. Early studies reveal that, like most eukaryotic proteins, the functions and activities of HDAC1 are regulated by post-translational modifications. For example, serine phosphorylation of HDAC1 by protein kinase CK2 promotes HDAC1 deacetylase enzymatic activity and alters its interactions with proteins in corepressor complexes. Here, we describe an alternative signaling pathway by which HDAC1 activities are regulated. Specifically, we discover that EGFR activity promotes the tyrosine phosphorylation of HDAC1, which is necessary for its protein stability. A key EGFR phosphorylation site on HDAC1, Tyr72, mediates HDAC1’s anti-apoptotic function. Given that HDAC1 overexpression and EGFR activity are strongly related with tumor progression and cancer cell survival, HDAC1 tyrosine phosphorylation may present a possible target to manipulate HDAC1 protein levels in future potential cancer treatment strategies.
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.
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