Kinase‐Catalyzed Crosslinking and Immunoprecipitation (K‐CLIP) to Explore Kinase‐Substrate Pairs

Abstract

Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase‐catalyzed crosslinking and immunoprecipitation (K‐CLIP). K‐CLIP uses a photocrosslinking γ‐phosphoryl‐modified ATP analog, such as ATP‐arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase‐substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K‐CLIP is an adaptable chemical tool to investigate and discover kinase‐substrate pairs, which will promote characterization of complex phosphorylation‐mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase‐catalyzed crosslinking of lysates Basic Protocol 2: Kinase‐catalyzed crosslinking and immunoprecipitation (K‐CLIP)

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

“…Only ATP-AFS 1d and ATP-HexBr 1e were successfully synthesized and purified, as described below. The ATP-aryl sulfonyl fluoride 1j was prone to hydrolysis and degraded during purification, ATP-propionyl bromide ( n = 2, 1g ) suffered from β-elimination of the bromide, and the other ATP-alkyl bromides either hydrolyzed ( n = 1, 1f ) or cyclized ( n = 3 and 4, 1h and 1i ) during purification, resulting in no desired product (data not shown) …”

“…The ATP-aryl sulfonyl fluoride 1j was prone to hydrolysis and degraded during purification, ATP-propionyl bromide (n = 2, 1g) suffered from β-elimination of the bromide, and the other ATP-alkyl bromides either hydrolyzed (n = 1, 1f) or cyclized (n = 3 and 4, 1h and 1i) during purification, resulting in no desired product (data not shown). 23 Synthesis of both ATP-AFS and ATP-HexBr began with the generation of the Boc-protected PEG linker (3), as previously published (Scheme S1). 24−26 27 After Boc deprotection under acidic conditions, the resulting amine was coupled to ATP at pH = 6.2−6.8 with Nmethylimidazole (NMI) 28 and EDCI to yield ATP-AFS (1d), which was characterized by NMR and HRMS (Figures S5− S10).…”

Valproate regulates inositol synthesis by reducing expression of myo-inositol-3-phosphate synthase

“…The second screen involved covalently crosslinking kinases to their substrates in a kinase-dependent manner, followed by identification of the crosslinked pairs by LC–MS/MS analysis. The kinase-catalyzed crosslinking and ion purification (K-CLiP) method 31 uses a γ-phosphoryl-modified adenosine 5’-triphosphate (ATP) analogue containing a photocrosslinking group to covalently link MIPS to both kinases and associated proteins. In K-CLiP, the ATP-photocrosslinker (ATP-PCL), ATP-arylazide, acts as a co-substrate of cellular kinases to transfer the arylazide photocrosslinker to 6xHis-Xpress-MIPS via kinase activity 32 .…”

Pharmacological approaches to understanding protein kinase signaling networks

“…Complexes containing crosslinked substrate and kinase can be purified by streptavidin pulldown or immunoprecipitation. Predicted KSRs can then be tested using western blots or, alternatively, unknown KSRs can be identified by proteomic analysis ( Dedigama-Arachchige and Pflum, 2016 ; Beltman and Pflum, 2022 ). Not all kinases can utilize unnatural ATP analogs to phosphorylate substrates and another disadvantage is that, because the ATP analogs do not cross cell membranes, these techniques cannot be performed in cells.…”