Development Of Kinase Catalyzed Biotinylation To Study Phosphoproteomics

Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to identify phosphoproteins in normal and diseased states is critical to fully characterize cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To explore kinase-catalyzed biotinylation, kinetic measurements were obtained with various kinases and the data indicated that kinase-catalyzed biotinylation occurs with catalytic efficiency appropriate for phosphoproteomics application. Next, the susceptibility of the biotin tag to phosphatases was characterized and found that the phosphorylbiotin group was relatively insensitive to protein phosphatases. Importantly, robust kinase-catalyzed biotinylation occurs without the need for phosphatase inhibitor treatment. The results suggest that kinase-catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low abundance phosphoproteins. Finally, we report application of kinase-catalyzed biotinylation to the enrichment of the phosphopeptides in cell lysates for mass spectrometry-based phosphoproteomics analysis. Significantly, the biotinylation strategy was used to enrich full-length phosphoprotein, which is challenging using metal ion purification. With the utility of ATP-biotin labeling for phosphoproteomics analysis established, kinase-catalyzed biotinylation can be applied to characterizing and understanding of the role of phosphorylation in various biological events

Structural Analysis of ATP Analogues Compatible with Kinase-Catalyzed Labeling

Kinase-catalyzed protein phosphorylation is an important biochemical process involved in cellular functions. We recently discovered that kinases promiscuously accept γ-modified ATP analogues as cosubstrates and used several ATP analogues as tools for studying protein phosphorylation. Herein, we explore the structural requirements of γ-modified ATP analogues for kinase compatibility. To understand the influence of linker length and composition, a series of ATP analogues was synthesized, and the efficiency of kinase-catalyzed labeling was determined by quantitative mass spectrometry. This study on factors influencing kinase cosubstrate promiscuity will enable design of ATP analogues for a variety of kinase-catalyzed labeling reactions

Methylation of dual-specificity phosphatase 4 controls cell differentiation

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS. [Display omitted] •Arginine methylation of DUSP4 by PRMT1 triggers HUWE1-mediated ubiquitylation•Megakaryocytes have heterogeneous PRMT1 expression levels•DUSP4 promotes megakaryocyte differentiation and polyploidization•Inhibition of PRMT1 or p38 kinase activities restores differentiation of MDS cells Su et al. report that methylation of DUSP4 by PRMT1 triggers DUSP4 ubiquitylation by HUWE1. DUSP4 inactivates p38 kinase to promote megakaryocyte differentiation. Abnormally high expression of PRMT1 and activation of p38 kinase in blood cells of MDS patients blocks megakaryopoiesis, revealing PRMT1 as a target for MDS treatment