Development Of Tools For Phosphosite-Specific Kinase Identification And Discovery Of Phosphatase Substrates

Phosphorylation is a ubiquitous post translational modification implicated in many diseases, such as cancer. The phosphorylation status of cellular proteins is regulated by the activity of kinases and phosphatases. The biological significance of many phosphorylation events remain unknown because the methods to determine which kinase or phosphatase is responsible for phosphorylation are limited. Previously, we established kinase-catalyzed labeling where kinases accept γ-modified ATP analogs, such as ATP-arylazide and ATP-biotin, to label phosphoproteins. To study substrates of kinases and phosphatases, here we developed two new methods using kinase-catalyzed labeling. As one application, we developed K-CLASP (Kinase-catalyzed CrossLinking And Streptavdin Purification) to identify the in-cellulo kinase of a phosphorylated site on a protein. In this case, we used ATP-arylazide to mediate crosslinking between a biotin tagged peptide carrying a phosphosite of interest and the respective kinase. Using Protein kinase A (PKA) and its known peptide substrate kemptide, we demonstrated that K-CLASP is capable of identifying PKA as the kinase responsible for kemptide phosphorylation in cell lysates. Then we used K-CLASP to identify the kinases that phosphorylate S178 of the Miz1 protein in a collaboration project. For phosphatase substrate identification, we developed K-BIPS (Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates). In prior work, we observed that labeling of phosphoproteins by ATP-biotin is reduced when phosphatases are inactive. The phosphatase dependency of biotinylation is due to the presence of already existing phosphorylation, which prevents ATP-biotin labeling. Therefore, in K-BIPS, ATP-biotin labeling is carried out after the inactivation of a particular phosphatase. The loss in biotinylation can then be analyzed to reveal substrates. To establish K-BIPS as a viable method, we carried out ATP-biotin labeling in lysates treated with the general phosphatase inhibitor okadaic acid. Many known phosphatase substrates were observed validating our method. Then as further applications, we used K-BIPS to explore substrates of PP1-Gadd34 and PP1-MYPT1 phosphatase complexes. The results demonstrate that K-BIS-Phos is a feasible method for phosphatase substrate identification. In summary, we have developed two chemical tools based on kinase-catalyzed labeling that will enhance our understanding of phosphorylation events mediated by kinases and phosphatases