Kinase-catalyzed Biotinylation for Discovery and Validation of Substrates to Multi-specificity Kinases NME1 and NME2.

Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression, but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multi-specificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS) method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the K-BILDS method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.

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. Here, kinetics and crosslinking experiments demonstrated that the UV-activated analogs, ATP-aryl azide and ATP-benzophenone, offered the most robust crosslinking, whereas electrophilic ATP-aryl fluorosulfate promoted the most effective proximity-enabled crosslinking. The data will guide future applications of kinase-catalyzed crosslinking to study normal and disease biology.

Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy.

CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)626 and Threonine (T)627 substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T627 is followed by a Proline (P), and this TP motif is conserved in vertebrates. Based on these findings, we generated affinity-purified, anti-phospho-CARP-1 T627 rabbit polyclonal antibodies, and utilized them to elucidate chemotherapy-activated, CARP-1-dependent cell growth signaling mechanisms. Our kinase profiling studies revealed that MAPKs/SAPKs phosphorylated CARP-1 T627. We then UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614-638) peptide. Our studies further established that SAPK p38γ, but not other MAPKs, phosphorylates CARP-1 T627 in cancer cells treated with genotoxic drugs. Loss of p38γ abrogates CARP-1 T627 phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. CARP-1 T627 phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T627 phosphorylation to inhibit cell growth.

Protein kinase A is a functional component of focal adhesions.

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify 53 high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3)-a well-established molecular scaffold, regulator of cell migration, and a component of focal and fibrillar adhesions-as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

Protein Kinase A is a Functional Component of Focal Adhesions.

Focal adhesions (FAs) form the junction between extracellular matrix (ECM)-bound integrins and the actin cytoskeleton and also transmit signals that regulate cell adhesion, cytoskeletal dynamics, and cell migration. While many of these signals are rooted in reversible tyrosine phosphorylation, phosphorylation of FA proteins on Ser/Thr residues is far more abundant yet its mechanisms and consequences are far less understood. The cAMP-dependent protein kinase (protein kinase A; PKA) has important roles in cell adhesion and cell migration and is both an effector and regulator of integrin-mediated adhesion to the ECM. Importantly, subcellular localization plays a critically important role in specifying PKA function. Here, we show that PKA is present in isolated FA-cytoskeleton complexes and active within FAs in live cells. Furthermore, using kinase-catalyzed biotinylation of isolated FA-cytoskeleton complexes, we identify fifty-three high-stringency candidate PKA substrates within FAs. From this list, we validate tensin-3 (Tns3) - a well-established molecular scaffold, regulator of cell migration, and component of focal and fibrillar adhesions - as a novel direct substrate for PKA. These observations identify a new pathway for phospho-regulation of Tns3 and, importantly, establish a new and important niche for localized PKA signaling and thus provide a foundation for further investigation of the role of PKA in the regulation of FA dynamics and signaling.

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. Despite their importance in cell biology, the role of phosphatases in cellular events has yet to be fully characterized, partly due to the lack of tools to identify phosphatase-substrate pairs in a biological context. The method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) was developed to remedy the lack of information surrounding phosphatase biology, particularly focused on substrate identification. In the K-BIPS method, the γ-phosphoryl modified adenosine 5'-triphosphate (ATP) analog, ATP-biotin, is used by kinases to biotin-label phosphoproteins. Because phosphatases must initially remove a phosphoryl group for subsequent biotinylation by ATP-biotin, phosphatase substrates are identified in K-BIPS by comparing biotinylated proteins in the presence and absence of active phosphatases. K-BIPS has been used to discover novel substrates of both serine/threonine and tyrosine phosphatases. This chapter describes the K-BIPS method to enable the identification of substrates to any phosphatases of interest, which will augment studies of phosphatase biology.

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. Here, the chemoproteomics method Kinase-catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS) was used to identify substrates of the PP1c-PPP1R12A complex in L6 skeletal muscle cells. K-BIPS enriched 136 candidate substrates with 14 high confidence hits. One high confidence hit, AKT1 kinase, was validated as a novel PP1c-PPP1R12A substrate. Given the previously documented role of AKT1 in PPP1R12A phosphorylation and cytoskeleton organization, the data suggest that PP1c-PPP1R12A regulates its own phosphatase activity through an AKT1-dependent feedback mechanism to influence cytoskeletal arrangement in muscle cells.

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

Inositol depletion is a hypothesized mechanism of action of mood stabilization drugs used in the treatment of bipolar disorder. It was previously reported that the mood stabilizer valproate (VPA) increased phosphorylation of myo-inositol-3-phosphate synthases (MIPS), the rate limiting enzyme of inositol synthesis. Phosphosites were identified and examination of site-directed mutants suggested that phosphorylation leads to decreased enzymatic activity. In this study, we examined the extent of MIPS phosphorylation in response to VPA and used two interaction screens to identify protein kinases that interact with MIPS. Using an epitope tagged MIPS construct, we determined the fraction of phosphorylated MIPS to be very low (less than 2% of total), and we could not detect phosphorylation of untagged MIPS in response to VPA. In vitro analyses of phosphorylation revealed that putative protein kinases, PKC and CKII, have low specificity toward MIPS. These findings suggest that VPA likely depletes inositol via a mechanism other than MIPS phosphorylation. Consistent with this, mRNA levels of the MIPS-encoding gene INO1 and MIPS protein levels were significantly reduced during the mid-log growth phase in response to VPA treatment. These findings suggest that the mechanism whereby VPA causes inositol depletion is by reducing expression of the rate-limiting enzyme MIPS.

Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS).

Protein phosphorylation is catalyzed by kinases to regulate a large variety of cellular activities, including growth and signal transduction. Methods to identify kinase substrates are crucial to fully understand phosphorylation-mediated cellular events and disease states. Here, we report a set of protocols to identify substrates of a target kinase using Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS). As described in these protocols, K-BILDS involves inactivation of endogenous kinases in lysates, followed by addition of an active exogenous kinase and the γ-phosphate-modified ATP analog ATP-biotin for kinase-catalyzed biotinylation of cellular substrates. Avidin enrichment isolates biotinylated substrates of the active kinase, which can be monitored by western blot. Substrates of the target kinase can also be discovered using mass spectrometry analysis. Key advantages of K-BILDS include compatibility with any lysate, tissue homogenate, or complex mixture of biological relevance and any active kinase of interest. K-BILDS is a versatile method for studying or discovering substrates of a kinase of interest to characterize biological pathways thoroughly. © 2023 Wiley Periodicals LLC. Basic Protocol 1: FSBA treatment of lysates to inactivate kinases Basic Protocol 2: Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS).

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. Because photocrosslinking ATP analogues require UV light, which could influence cell biology, we report here two ATP analogues, ATP-aryl fluorosulfate (ATP-AFS) and ATP-hexanoyl bromide (ATP-HexBr), that crosslink kinase-substrate pairs via proximity-mediated reactions without the need for UV irradiation. Both ATP-AFS and ATP-HexBr acted as cosubstrates with a variety of kinases for affinity-based crosslinking, with ATP-AFS showing more robust complexes. Importantly, ATP-AFS promoted crosslinking in lysates, which demonstrates compatibility with complex cellular mixtures for future application to kinase-substrate identification.

Proteomics-based trapping with single or multiple inactive mutants reproducibly profiles histone deacetylase 1 substrates.

Histone deacetylase 1 (HDAC1) plays a key role in diverse cellular processes. With the aberrant expression of HDAC1 linked to many diseases, including cancers, HDAC inhibitors have been used successfully as therapeutics. HDAC1 has been predominantly associated with histone deacetylation and gene expression. Recently, non-histone substrates have revealed diverse roles of HDAC1 beyond epigenetics. To augment discovery of non-histone substrates, we introduced "substrate trapping" to enrich HDAC1 substrates using an inactive mutant. Herein, we performed a series of proteomics studies to test the robustness of HDAC1 substrate trapping. Based on our recent results documenting that different HDAC1 mutants preferentially bound different substrates, which suggested that multiple mutants could be used for efficient trapping, trapping with three single point mutants simultaneously identified several potential substrates uniquely compared to a single mutant alone. However, a greater number of biologically interesting hits were observed using only a single mutant, which suggests that the C151A HDAC1 mutant is the optimal trap. Importantly, comparing independent trials with a single mutant performed by different experimentalists and HEK293 cell populations, trapping was robust and reproducible. Based on the reproducible trapping data, carnosine N-methyltransferase 1 (CARNMT1) was validated as an HDAC1 substrate. The data document that mutant trapping is an effective method for discovery of unanticipated HDAC substrates. SIGNIFICANCE: Histone deacetylase (HDAC) proteins are well established epigenetic transcriptional regulators that deacetylate histone substrates to control gene expression. More recently, deacetylation of non-histone substrates has linked HDAC activity to functions outside of epigenetics. Given the use of HDAC inhibitor drugs as anti-cancer therapeutics, understanding the full functions of HDAC proteins in cell biology is essential to future drug design. To discover unanticipated non-histone substrates and further characterize HDAC functions, inactive mutants have been used to "trap" putative substrates, which were identified with mass spectrometry-based proteomics analysis. Here multiple trapping studies were performed to test the robustness of using inactive mutants and proteomics for HDAC substrate discovery. The data confirm the value of trapping mutants as effective tools to discover HDAC substrates and link HDAC activity to unexpected biological functions.

First-in-Class Dual Mechanism Ferroptosis-HDAC Inhibitor Hybrids.

HDAC inhibitors are an attractive class of cytotoxic agents for the design of hybrid molecules. Several HDAC hybrids have emerged over the years, but none combines HDAC inhibition with ferroptosis, a combination which is being extensively studied because it leads to enhanced cytotoxicity and attenuated neuronal toxicity. We combined the pharmacophores of SAHA and CETZOLE molecules to design the first-in-class dual mechanism hybrid molecules, which induce ferroptosis and inhibit HDAC proteins. The involvement of both mechanisms in cytotoxicity was confirmed by a series of biological assays. The cytotoxic effects were evaluated in a series of cancer and neuronal cell lines. Analogue HY-1 demonstrated the best cytotoxic profile with GI50 values as low as 20 nM. Although the increase in activity of the hybrids over the combinations is modest in cellular systems, they have the potential advantage of homogeneous spatiotemporal distribution in in vivo systems.

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

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).

Evidence that HDAC7 acts as an epigenetic "reader" of AR acetylation through NCoR-HDAC3 dissociation.

Histone deacetylase (HDAC) proteins are epigenetic regulators that govern a wide variety of cellular events. With a role in cancer formation, HDAC inhibitors have emerged as anti-cancer therapeutics. Among the eleven metal-dependent class I, II, and IV HDAC proteins targeted by inhibitor drugs, class IIa HDAC4, -5, -7, and -9 harbor low deacetylase activity and are hypothesized to be "reader" proteins, which bind to post-translationally acetylated lysine. However, evidence linking acetyllysine binding to a downstream functional event is lacking. Here, we report for the first time that HDAC4, -5, and -7 dissociated from corepressor NCoR in the presence of an acetyllysine-containing peptide, consistent with reader function. Documenting the biological consequences of this possible reader function, mutation of a critical acetylation site regulated androgen receptor (AR) transcriptional activation function through HDAC7-NCoR-HDAC3 dissociation. The data document the first evidence consistent with epigenetic-reader functions of class IIa HDAC proteins.

Kinase-Catalyzed Biotinylation to Map Cell Signaling Pathways: Application to Epidermal Growth Factor Signaling.

Cell signaling involves a network of protein-protein interactions and post-translational modifications that govern cellular responses to environmental cues. To understand and ultimately modulate these signaling pathways to confront disease, the complex web of proteins that becomes phosphorylated after extracellular stimulation has been studied using mass spectrometry-based proteomics methods. To complement prior work and fully characterize all phosphorylated proteins after the stimulation of cell signaling, we developed K-BMAPS (kinase-catalyzed biotinylation to map signaling), which utilizes ATP-biotin as a kinase cosubstrate to biotin label substrates. As a first application of the K-BMAPS method, the well-characterized epidermal growth factor receptor (EGFR) kinase signaling pathway was monitored by treating epidermal growth factor (EGF)-stimulated HeLa lysates with ATP-biotin, followed by streptavidin enrichment and quantitative mass spectrometry analysis. On the basis of the dynamic phosphoproteins identified, a pathway map was developed considering functional categories and known interactors of EGFR. Remarkably, 94% of the K-BMAPS hit proteins were included in the EGFR pathway map. With many proteins involved in transcription, translation, cell adhesion, and GTPase signaling, K-BMAPS identified phosphoproteins were associated with late and continuous signaling events. In summary, the K-BMAPS method is a powerful tool to map the dynamic phosphorylation governing cell signaling pathways.

A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics.

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC50 values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI50 values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

HDAC6 Substrate Discovery Using Proteomics-Based Substrate Trapping: HDAC6 Deacetylates PRMT5 to Influence Methyltransferase Activity.

Histone deacetylase 6 (HDAC6) is upregulated in a variety of tumor cell lines and has been linked to many cellular processes, such as cell signaling, protein degradation, cell survival, and cell motility. HDAC6 is an enzyme that deacetylates the acetyllysine residues of protein substrates, and the discovery of HDAC6 substrates, including tubulin, has revealed many roles of HDAC6 in cell biology. Unfortunately, among the wide variety of acetylated proteins in the cell, only a few are verified as HDAC6 substrates, which limits the full characterization of HDAC6 cellular functions. Substrate trapping mutants were recently established as a tool to discover unanticipated substrates of histone deacetylase 1 (HDAC1). In this study, we applied the trapping approach to identify potential HDAC6 substrates. Among the high confidence protein hits after trapping, protein arginine methyl transferase 5 (PRMT5) was successfully validated as a novel HDAC6 substrate. PRMT5 acetylation enhanced its methyltransferase activity and symmetrical dimethylation of downstream substrates, revealing possible crosstalk between acetylation and methylation. Substrate trapping represents a powerful, systematic, and unbiased approach to discover substrates of HDAC6.

EGFR phosphorylates HDAC1 to regulate its expression and anti-apoptotic function.

HDAC1 is the prototypical human histone deacetylase (HDAC) enzyme responsible for catalyzing the removal of acetyl group from lysine residues on many substrate proteins. By deacetylating histones and non-histone proteins, HDAC1 has a profound effect on the regulation of gene transcription and many processes related to cell growth and cell death, including cell cycle progression, DNA repair, and apoptosis. Early studies reveal that, like most eukaryotic proteins, the functions and activities of HDAC1 are regulated by post-translational modifications. For example, serine phosphorylation of HDAC1 by protein kinase CK2 promotes HDAC1 deacetylase enzymatic activity and alters its interactions with proteins in corepressor complexes. Here, we describe an alternative signaling pathway by which HDAC1 activities are regulated. Specifically, we discover that EGFR activity promotes the tyrosine phosphorylation of HDAC1, which is necessary for its protein stability. A key EGFR phosphorylation site on HDAC1, Tyr72, mediates HDAC1's anti-apoptotic function. Given that HDAC1 overexpression and EGFR activity are strongly related with tumor progression and cancer cell survival, HDAC1 tyrosine phosphorylation may present a possible target to manipulate HDAC1 protein levels in future potential cancer treatment strategies.

Differential profiles of HDAC1 substrates and associated proteins in breast cancer cells revealed by trapping.

Histone deacetylase (HDAC) proteins, which regulate the acetylation state of proteins, are the targets of multiple clinical drugs for cancer treatment. Due to the heterogeneity of tumors, HDAC proteins play different roles in the progression of various cancer types. For example, MDA-MB-468 and MDA-MB-231 cells are both triple negative breast cancer cells but belong to different subtypes that display different response to HDAC inhibitor drugs. To investigate the role of HDAC proteins in breast cancer, the substrate and associated proteins of HDAC1 in MDA-MB-231, MDA-MB-468, and a normal breast epithelial cell line, MCF10A, were analyzed using substrate trapping mutants and proteomics-based mass spectrometry. All three cell lines demonstrated nonoverlapping substrate protein profiles. While both normal MCF10A and cancerous MDA-MB-468 cell lines contained similar HDAC1 associated proteins, including proteins associated with epigenetic and RNA processing mechanisms, the HDAC1 associated protein profile of MDA-MB-231 cells was devoid of expected epigenetic proteins. The variable associated protein profiles of MDA-MB-231 and MDA-MB-468 suggest that HDAC1 plays distinct roles in breast cancer cell biology, which might affect cancer aggressiveness and HDAC inhibitor sensitivity.

An Affinity-Based, Cysteine-Specific ATP Analog for Kinase-Catalyzed Crosslinking.

Kinases mediate cell signaling pathways by catalyzing protein phosphorylation. Irregularities in kinase activity are directly associated with disease conditions. Therefore, methods to identify substrates of a particular kinase are needed to understand signaling cascades in normal and diseased states. Photocrosslinking ATP analogs provide powerful tools to study kinases by covalently linking kinases with substrates. However, the involvement of UV light and nonspecific reactivity of current ATP-photocrosslinkers challenge kinase-substrate identification. We report here an affinity-based crosslinking ATP analog, ATP-methylacrylamide (ATP-MAc), that contains a cysteine-reactive acrylamide crosslinking group, which avoids the UV irradiation and non-specific reactivity of prior analogs. Using in vitro kinase assays, ATP-MAc acts as a kinase co-substrate and covalently crosslinks only kinases containing cysteines in the active site. ATP-MAc was also able to crosslink cellular proteins in lysates, documenting compatibility with cell-based studies.