Kinase-catalyzed crosslinking: A comparison of ATP-crosslinker analogs.

Protein phosphorylation is catalyzed by kinases to regulate cellular events and disease states. Identifying kinase-substrate relationships represents a powerful strategy to understand cell biology and disease yet remains challenging due to the rapid dynamics of phosphorylation. Over the last decade, several γ-phosphoryl modified ATP analogs containing crosslinkers were developed to covalently conjugate kinases, their substrates, and their associated proteins for subsequent characterization.

Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS).

Phosphorylation is a reversible post-translational modification that alters the functions of proteins to govern various cellular events, including cell signaling. Kinases catalyze the transfer of a phosphoryl group onto the hydroxyl residue of serine, threonine, and tyrosine, while phosphatases catalyze the removal. Unregulated kinase and phosphatase activity have been observed in various cancers and neurodegenerative diseases.

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

Protein phosphatase 1 regulatory subunit 12A (PPP1R12A) interacts with the catalytic subunit of protein phosphatase 1 (PP1c) to form the myosin phosphatase complex. In addition to a well-documented role in muscle contraction, the PP1c-PPP1R12A complex is associated with cytoskeleton organization, cell migration and adhesion, and insulin signaling. Despite the variety of biological functions, only a few substrates of the PP1c-PPP1R12A complex are characterized, which limit a full understanding of PP1c-PPP1R12A activities in muscle contraction and cytoskeleton regulation.

Affinity-Based Kinase-Catalyzed Crosslinking to Study Kinase-Substrate Pairs.

Phosphorylation of proteins by kinase enzymes is a post-translational modification involved in a myriad of biological events, including cell signaling and disease development. Identifying the interactions between a kinase and its phosphorylated substrate(s) is necessary to characterize phosphorylation-mediated cellular events and encourage development of kinase-targeting drugs. One method for substrate-kinase identification utilizes photocrosslinking γ-phosphate-modified ATP analogues to covalently link kinases to their substrates for subsequent monitoring.