Identifying specific kinase substrates through engineered kinases and ATP analogs

Intracellular signaling by protein kinases controls many aspects of cellular biochemistry and physiology. Determining the direct substrates of protein kinases is important in understanding how these signaling enzymes exert their effect on cellular functions. One of the recent developments in this area takes advantage of the similarity in the ATP binding domains of protein kinases, where a few conserved amino acids containing large side chains come in close contact with the N-6 position of bound ATP. Mutation of one or more of these residues generates a "pocket" in the ATP binding site that allows the mutant kinase, but not other cellular kinases, to utilize analogs of ATP with bulky substituents synthesized onto the N-6 position. The use of such a mutated kinase and radiolabeled ATP analogs allows for the specific labeling of direct substrates of the kinase within a mixture of cellular proteins. We have recently reported the generation of "pocket" mutants of extracellular regulated kinase 2 (ERK2) and their use in the identification of two novel substrates of ERK2. In this report, we discuss the generation and characterization of ERK2 mutants that utilize analogs of ATP and describe the methodology used to identify ERK2-associated substrates. We also describe the direct labeling of ERK2 substrates in cell lysates. These methodologies can be adapted for use with other protein kinases to increase the understanding of intracellular signal transduction.     

Engineering the serine/threonine protein kinase Raf-1 to utilise an orthogonal analogue of ATP substituted at the N6 position

One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild-type enzyme and the majority of other kinases. The acceptance of structurally modified, gamma-(32)P-labelled, nucleotide analogue by active site-modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf-1, which plays an essential role in the transduction of stimuli through the Ras-->Raf-->MEK-->ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf-1, such that it can utilise an N(6)-substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf-1. We screen a library of analogues to identify which are orthogonal for Raf-1, and show that mutant Raf-1 can utilise the orthogonal analogue N(6)(2-phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf-1 kinase with (32)P-N(6)(2-phenethyl) ATP in cell lysates.     

QIKS – Quantitative identification of kinase substrates

Signaling networks regulate cellular responses to external stimuli through post‐translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large‐scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel‐screening platform developed for the proteome‐wide substrate‐analysis of specific kinases. Here, we aimed to identify substrates of mitogen‐activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen‐activated protein kinase cascade. An ATP analog‐sensitive mutant of Mek1 (Mek1‐as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC‐MS/MS of IMAC‐enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.     

Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway

The mammalian dual-specificity protein-tyrosine phosphatase VHR (for VH1-related) has been identified as a novel regulator of extracellular regulated kinases (ERKs). To identify potential cellular substrates of VHR, covalently immobilized mutant VHR protein was employed as an affinity trap. A tyrosine-phosphorylated protein(s) of approximately 42 kDa was specifically adsorbed by the affinity column and identified as ERK1 and ERK2. Subsequent kinetic analyses and transfection studies demonstrated that VHR specifically dephosphorylates and inactivates ERK1 and ERK2 in vitro and in vivo. Only the native structure of phosphorylated ERK was recognized by VHR and was inactivated with a second-order rate constant of 40,000 M-1 s-1. VHR was found to dephosphorylate endogenous ERK, but not p38 and JNK. Immunodepletion of endogenous VHR eliminated the dephosphorylation of cellular ERK. Transfection studies in COS-1 cells demonstrated that in vivo phosphorylation of epidermal growth factor-stimulated ERK depended on VHR protein levels. Overexpression above endogenous levels of VHR led to accelerated ERK inactivation, but did not alter the normal activation of ERK. Unique among reported mitogen activated protein kinase phosphatases, VHR is constitutively expressed, localized to the nucleus, and tyrosine-specific. This study is the first to report the identification of authentic substrates of dual-specificity phosphatases utilizing affinity absorbents and is the first to identify a nuclear, constitutively expressed, and tyrosine-specific ERK phosphatase. The data strongly suggest that VHR is responsible for the rapid inactivation of ERK following stimulation and for its repression in quiescent cells.     

Phosphorylation by LAMMER protein kinases: determination of a consensus site, identification of in vitro substrates, and implications for substrate preferences

LAMMER protein kinases are ubiquitous throughout eukaryotes, including multiple paralogues in mammals. Members are characterized by similar overall structure and highly identical amino acid sequence motifs in catalytic subdomains essential for phosphotransfer and interaction with substrates. LAMMER kinases phosphorylate and regulate the activity of the SR protein class of pre-mRNA splicing components, both in vitro and in vivo. In this study, we define an optimum in vitro consensus phosphorylation site for three family members using an oriented degenerate peptide library approach. We also examine the substrate specificity and interactions of several LAMMER protein kinases from widely diverged species with potential substrates, including their own N-termini, predicted to be substrates by the peptide-based approach. Although the optimal in vitro consensus phosphorylation site for these kinases is remarkably similar for short peptides, distinct substrate preferences are revealed by in vitro phosphorylation of intact proteins. This finding suggests that these kinases may possess varied substrates in vivo, and thus the multiple LAMMER kinases present in higher eukaryotes may perform differentiable functions. These results further demonstrate that these kinases can phosphorylate a number of substrates in addition to SR proteins, suggesting that they may regulate multiple cellular processes, in addition to the alternative splicing of pre-mRNAs.     

Experimental validation of the docking orientation of Cdc25 with its Cdk2-CycA protein substrate

Cdc25 phosphatases are key activators of the eukaryotic cell cycle and compelling anticancer targets because their overexpression has been associated with numerous cancers. However, drug discovery targeting these phosphatases has been hampered by the lack of structural information about how Cdc25s interact with their native protein substrates, the cyclin-dependent kinases. Herein, we predict a docked orientation for Cdc25B with its Cdk2-pTpY-CycA protein substrate by a rigid-body docking method and refine the docked models with full-scale molecular dynamics simulations and minimization. We validate the stable ensemble structure experimentally by a variety of in vitro and in vivo techniques. Specifically, we compare our model with a crystal structure of the substrate-trapping mutant of Cdc25B. We identify and validate in vivo a novel hot-spot residue on Cdc25B (Arg492) that plays a central role in protein substrate recognition. We identify a hot-spot residue on the substrate Cdk2 (Asp206) and confirm its interaction with hot-spot residues on Cdc25 using hot-spot swapping and double mutant cycles to derive interaction energies. Our experimentally validated model is consistent with previous studies of Cdk2 and its interaction partners and initiates the opportunity for drug discovery of inhibitors that target the remote binding sites of this protein-protein interaction.     

Phosphoproteomic identification and functional characterization of protein kinase substrates by 2D-DIGE and Phos-tag PAGE

Protein phosphorylation is one of the most common post-translational modifications in eukaryotes and can regulate diverse properties of proteins. Protein kinases are encoded by more than 500 genes in higher eukaryotes and play central roles in various cellular signaling pathways. Consequently, genetic abnormalities of protein kinases have been implicated in many diseases. To fully understand the complex phosphorylation-mediated signaling networks, it is important to globally identify and functionally characterize in vivo substrates of individual protein kinases. Advances in electrophoresis-based phosphoproteomic technologies such as two-dimensional difference gel electrophoresis (2D-DIGE) following immobilized metal affinity chromatography (IMAC) and phosphate-affinity Phos-tag PAGE have enabled efficient and detailed analysis of protein kinase substrates. Here, we describe physiological functions of the newly identified substrates of several disease-related protein kinases including ERK, PKD and PINK1.     

Identification of ChChd3 as a novel substrate of the cAMP-dependent protein kinase (PKA) using an analog-sensitive catalytic subunit

Due to the numerous kinases in the cell, many with overlapping substrates, it is difficult to find novel substrates for a specific kinase. To identify novel substrates of cAMP-dependent protein kinase (PKA), the PKA catalytic subunit was engineered to accept bulky N(6)-substituted ATP analogs, using a chemical genetics approach initially pioneered with v-Src (1). Methionine 120 was mutated to glycine in the ATP-binding pocket of the catalytic subunit. To express the stable mutant C-subunit in Escherichia coli required co-expression with PDK1. This mutant protein was active and fully phosphorylated on Thr(197) and Ser(338). Based on its kinetic properties, the engineered C-subunit preferred N(6)(benzyl)-ATP and N(6)(phenethyl)-ATP over other ATP analogs, but still retained a 30 microm K(m) for ATP. This mutant recombinant C-subunit was used to identify three novel PKA substrates. One protein, a novel mitochondrial ChChd protein, ChChd3, was identified, suggesting that PKA may regulate mitochondria proteins.     

Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation

The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIalpha, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIalpha in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.     

Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry

Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.     

The chaperone function of DnaK requires the coupling of ATPase activity with substrate binding through residue E171.

Central to the chaperone function of Hsp70 stress proteins including Escherichia coli DnaK is the ability of Hsp70 to bind unfolded protein substrates in an ATP-dependent manner. Mg2+/ATP dissociates bound substrates and, furthermore, substrate binding stimulates the ATPase of Hsp70. This coupling is proposed to require a glutamate residue, E175 of bovine Hsc70, that is entirely conserved within the Hsp70 family, as it contacts bound Mg2+/ATP and is part of a hinge required for a postulated ATP-dependent opening/closing movement of the nucleotide binding cleft which then triggers substrate release. We analyzed the effects of dnaK mutations which alter the corresponding glutamate-171 of DnaK to alanine, leucine or lysine. In vivo, the mutated dnaK alleles failed to complement the delta dnaK52 mutation and were dominant negative in dnaK+ cells. In vitro, all three mutant DnaK proteins were inactive in known DnaK-dependent reactions, including refolding of denatured luciferase and initiation of lambda DNA replication. The mutant proteins retained ATPase activity, as well as the capacity to bind peptide substrates. The intrinsic ATPase activities of the mutant proteins, however, did exhibit increased Km and Vmax values. More importantly, these mutant proteins showed no stimulation of ATPase activity by substrates and no substrate dissociation by Mg2+/ATP. Thus, glutamate-171 is required for coupling of ATPase activity with substrate binding, and this coupling is essential for the chaperone function of DnaK.     

GRK2-dependent desensitization downstream of G proteins

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of beta-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/-) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.     

Substrate specificities and identification of putative substrates of ATM kinase family members

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.     

In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.     

GRK2-Dependent Desensitization Downstream of G Proteins

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of β-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/?) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.     

Calcium activation of ERK mediated by calmodulin kinase I

Elevated intracellular Ca(2+) triggers numerous signaling pathways including protein kinases such as the calmodulin-dependent kinases (CaMKs) and the extracellular signal-regulated kinases (ERKs). In the present study we examined Ca(2+)-dependent "cross-talk" between these two protein kinase families. Using a combination of pharmacological inhibitors and dominant-negative kinases (dnKinase), we identified a requirement for CaMKK acting through CaMKI in the stimulation of ERKs upon depolarization of the neuroblastoma cell line, NG108. Depolarization stimulated prolonged ERK and JNK activation that was blocked by the CaMKK inhibitor, STO-609; this inhibition of ERK activation by STO-609 was rescued by expression of a STO-609-insensitive mutant of CaMKK. However, activation of ERK by epidermal growth factor or carbachol were not suppressed by inhibition of CaMKK, indicating specificity for this "cross-talk." To identify the downstream target of CaMKK that mediated ERK activation upon depolarization, dnKinases were expressed. The dnCaMKI completely suppressed ERK2 activation whereas dnAKT/PKB or nuclear-targeted dnCaMKIV, other substrates for CaMKK, were not inhibitory. ERK activation upon depolarization or transfection with constitutively active (ca) CaMKI was blocked by dnRas. Additionally, depolarization of NG108 cells promoted neurite outgrowth, and this effect was blocked by inhibition of either CaMKK (STO-609) or ERK (UO126). Co-transfection with caCaMKK plus caCaMKI also stimulated neurite outgrowth that was blocked by inhibition of ERK (UO126). These data are the first to suggest that ERK activation and neurite outgrowth in response to depolarization are mediated by CaMKK activation of CaMKI.