The generality of kinase-catalyzed biotinylation

Abstract

Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signalling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.

K‐BILDS: A Kinase Substrate Discovery Tool

“…Moreover, thorough in cellulo or in vivo studies are needed to explore the physiological relevance of PKA phosphorylation on the function of identified substrates. Given the general ability of kinases to accept ATP‐biotin as a cosubstrate, K‐BILDS represents an enabling tool to identify substrates of any protein kinase.…”

“…In previous work, our laboratory and others discovered that a variety of γ‐phosphate‐modified ATP analogues act as kinase cosubstrates . Specifically, ATP modified with a biotin tag at γ‐phosphate acts as a kinase cosubstrate to transfer a phosphoramidyl biotin group onto protein substrates (Figure A) . Kinase‐catalyzed biotinylation has been used to visualize proteins but has not yet been exploited in kinase substrate identification.…”

“…The advantages of K‐BILDS include the generalizability to all kinases and substrates, the strong biotin–streptavidin interaction for purification, and the use of highly sensitive LC‐MS/MS for substrate detection. To establish K‐BILDS as a substrate identification tool, we focused on a well‐studied kinase, cAMP‐dependent protein kinase A (PKA/PRKACA).…”

Identifying Unknown Enzyme–Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry

“…Screening is a common approach for identifying enzyme–substrate pairs . For enzyme families with common substrates, such as S ‐adenosyl‐ l ‐methionine (AdoMet or SAM) for methyltransferases or adenosine triphosphate (ATP) for kinases, radiolabeled or affinity‐labeled substrates, such as biotin‐labeled ATP, or bio‐orthogonally tagged analogues, such as propargyl and ketone AdoMet analogues, have been used to screen for enzyme–substrate pairs . However, this methodology fails to identify specific enzyme–substrate pairs, particularly from cellular contexts, as multiple substrates can be tagged by multiple enzymes with no clear path for deconvolution.…”

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

“…For example, kinases collaborate with ATP-biotin ( Figure 1A) to attach a phosphoryl biotin tag to substrates, facilitating protein visualization and purification. Importantly for K-BIPS, the extent of protein biotinylation by ATP-biotin in lysates was reduced significantly in the presence of phosphatase inhibitors, [21][22][23][24] indicating that active phosphatases are required for full kinase-catalyzed biotinylation of cellular proteins. Based on this phosphatase dependence, K-BIPS compares the relative biotinylation of proteins in untreated and phosphatase-inactivated cell lysates after the ATP-biotin reaction.…”