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Protein kinases are important regulators of cellular function, and the dynamics of their activities are critical indicators of the health or pathology of living systems.[1, 2] In particular, extracellular-signal regulated kinases 1 and 2 (ERK1/2) play a pivotal role in the mitogen-activated protein kinase (MAPK) signaling pathway responsible for regulated cell survival and proliferation. The centrality of these enzymes in normal and diseased cell states underscores the need for high throughput, selective, and sensitive methods that accurately and directly diagnose kinase activities. The benchmark phosphorylation assays for ERK1/2 rely on transfer of radioactive γ-phosphate of [γ-32P]ATP to peptide or protein substrates. While broadly employed, this approach has limitations, including the discontinuous nature of the radioactive assay and the non-native ATP concentrations that are employed. Alternatively, for cellular imaging, genetically-encoded sensors that rely on phosphorylation-based changes in fluorescence resonance energy transfer (FRET) between fluorescent protein pairs[5, 6] have been constructed for several kinases, including ERK1/2.[7–10] These sensors are powerful because they can be expressed in cells, however, they cannot be used for high throuput screening of recombinant enzymes and unfractionated cell lysates due to the very limited fluorescence changes that accompany phosphorylation. As a complementary approach, probes based on small, organic fluorophores with direct readouts[6, 11] can give sensitive and robust signals under physiogical conditions and are thus amenable to high throughput applications. For example, we have incorporated a sulfonamido-oxine (Sox) chromophore into peptides[12, 13] to report phosphorylation via chelation-enhanced fluorescence (CHEF) (Figure 1a). The weak binding affinity of the unphosphorylated substrate for Mg2+ increases significantly upon phosphorylation, resulting in robust (2- to 12-fold) fluorescence enhancements. This versatile peptide-based sensor design has been applied to monitor the activity of numerous Ser/Thr and Tyr kinases both in vitro and in cell lysates.
With more than 500 different kinases encoded in the human genome, sensor selectivity becomes paramount, particularly when studying enzymes under conditions that resemble their native environments, such as in unfractionated cell lysates or live cells. While many protein kinases drive specificity from linear recognition motifs comprising 4–8 residues that are proximal to the phosphorylation site, a number of physiologically important kinases, including ERK1/2 and other MAPKs, phosphorylate substrates with short and ubiquitous consensus sequences. For these kinases, specificity is derived from extended recognition elements that include protein-protein interactions distal to the phosphorylation site. For example, ERK1 and 2 phosphorylate the transcription factor Ets-1 at Thr38 within a short ThrPro (TP) consensus motif. Since this short sequence would be the target of multiple kinases, ERK recognition of Ets-1 depends an adjoining N-terminal pointed (PNT) domain to dock the substrate specifically to ERK1/2, which engages the phosphorylation machinery. With this docking-domain strategy, PNT-based substrates demonstrate good affinity for ERK1/2 (KM ~ 6–9 μM), in stark contrast to short peptide substrates derived only from the TP sequence (KM > 200 μM).[15, 17] Due to the size of the PNT domain (11 kDa) these docking-domain interactions cannot be exploited with the types of synthetic peptide-based sensors that have been previously reported. However, in light of its importance in cellular homeostasis and the prominence of ERK disregulation in cancer, we set out to construct a chimeric sensor for ERK1/2 that combines the advantageous reporting properties of the Sox fluorophore with the outstanding specificity provided by the native protein domain-based recognition (Figure 1a). Most importantly, to ensure facile and high throughput analysis of ERK1/2 activity, our ultimate requirement is that the probe be highly selective in cell lysates where it would be exposed to hundreds of other active kinases.
Herein we describe the semisynthesis of a chimeric Sox-based ERK1/2 sensor through a key native chemical ligation (NCL) reaction that efficiently conjugates the recombinat PNT domain of Ets-1 to a synthetic ERK1/2 consensus sequence including the Sox sensing module. The extended PNT recognition element confers the ERK1/2 sensor with excellent selectivity, as demonstrated by comparative quantitative analyses with a panel of related recombinant enzymes and in unfractionated lysates from four different cell lines. Most importantly, the docking domain-based sensor design should be generally applicable to the development of selective sensors for other medically important kinases.
The new sensor was assembled as illustrated in Figure 1, using NCL, to ligate the synthetic Sox-containing peptide thioester with the expressed PNT domain, comprising Ets-1 residues 46–138 (Figure 1b). The peptide thioester was synthesized using Fmoc-based solid phase peptide synthesis (SPPS) on highly acid-labile TGT resin, followed by an off-bead thioesterification of the protected peptide. The Sox chromophore was introduced as the amino acid C-Sox. An optimized phosphorylation motif based on the ERK2 phosphorylation sequence within the myelin basic protein (MBP) (TPGGRR) was used in place of the phosphorylated region of Ets-1 (TPSSKE) to improve fluorescent properties of peptidyl probes (Table S1 in the Supporting Information). The distance between the TP recognition sequon and the PNT domain in the wild type protein was preserved in the sensor (Table S2 in the Supporting Information). This design introduced residue replacements in the unstructured N-terminal region of Ets-1, thereby minimizing perturbations to the overall secondary structure. Additionally, the C-terminal residue, Met44, was changed to Gly to eliminate the possibility of epimerization during thioesterification and to increase ligation efficiency. The expressed C-terminal fragment of the sensor, GST-PNT, was proteolyzed to reveal Cys-PNT. After ligation of Cys-PNT to the peptide thioester in non-denaturing conditions, the ERK1/2 probe, Sox-PNT, was obtained with excellent conversion (80–90%) (Figure S1 in the Supporting Information). The corresponding phosphoprotein (pThr38), pSox-PNT, was constructed using analogous methods.
Initial spectroscopic studies with Sox-PNT and pSox-PNT revealed a robust 3-fold enhancement in fluorescence on phosphorylation (Figure S2 in the Supporting Information). Subsequent in vitro assays determined Sox-PNT to be an efficient substrate for ERK2 when compared with the corresponding Sox-peptide (Ac-VP-CSox-LTPGGRRG-OH) (Figure 2a and Figure S3 in the Supporting Information). Furthermore, Sox-PNT demonstrated a KM of 14.9 μM for ERK2, which is comparable to the reported value for wild type PNT (9 μM), indicating that use of the MBP TP sequence has minimal effect relative to the native protein (Figure 2b). In contrast, the MBPtide (APRTPGGRR), the basis of the Sox-PNT phosphorylation sequence, was reported to have a KM of 2 mM for ERK2, underscoring the importance of the PNT domain in substrate kinetics. Finally, Sox-PNT exhibited high selectivity for ERK1/2 when compared to related kinases from the JNK, p38 and CDK families (Figure 2c).
In order to demonstrate that the preference of Sox-PNT for ERK1/2 can translate to complex media, the probe was exposed to a panel of unfractionated cell lysates that contained varying levels of active ERK1/2. The activity of cellular ERK1/2 is linked to its phosphorylation state, which is modulated by the epidermal growth factor (EGF) signaling pathway (Figure 3a). In summary, EGF interacts with EGF receptors (EGFRs), which leads to activation of MEK1/2, which in turn phosphorylates and activates ERK1/2. This event can be regulated either by the upstream MEK1/2 inhibitor, U0126, or by a direct ERK1/2 inhibitor, PEA-15. Summarized in Figure 3b are the results of ERK1/2 activity analyses on the crude lysates from four mammalian cell lines, which reveal the selectivity of the sensor in these complex media. In all cases, untreated lysates showed relatively low basal activity. Upon EGF stimulation, there was a 4- to 10-fold increase in activity. To demonstrate that Sox-PNT was specifically monitoring ERK1/2 activity, cells were exposed to the inhibitor U0126 and subsequently EGF-stimulated and lysed. Under these conditions, the ERK1/2 activity was returned to nearly basal levels. Western blot analysis was used to demonstrate that both stimulated and U0126 inhibitor-treated cells expressed ERK1/2 (Figure 3b), however, only EGF-treated samples showed enhanced levels of activated ERK1/2 as evidenced by analysis with the phopho-ERK1/2-specific antibody. In contrast, the Sox-peptide, lacking the PNT docking domain, showed promiscuous activity signaling phosphorylation that could not be correlated with ERK1/2 activity (Figure 3c).
Further evidence that Sox-PNT is selectively modified by ERK1/2 was obtained with PEA-15, a direct protein inhibitor of ERK1/2. Titration of PEA-15 into EGF-stimulated NIH-3T3 lysates created a dose-dependent response with a half inhibitory concentration of 40 nM and a Ki (30 nM) that reflected the reported Ki values (20 nM) (Figure 3d and the Supporting Information). To directly correlate the observed fluorescent signal to the presence of ERK, we exposed our probe to ERK1/2-depleted lysates. Indeed, immunodepletion of ERK1/2 from EGF-stimulated HeLa lysate reduced activity by 7-fold compared to the input lysate or the sample that had been depleted with naïve rabbit IgG (Figure 3e). This indicates that the Sox-PNT signal is predominantly due to the ERK1/2-mediated phosphorylation. Immunodepletion was confirmed by western blot analysis with the immunodepleting antibody (Figure 3e, inset). Having validated the selectivity of Sox-PNT for ERK1/2, the sensor can be used to measure active ERK1/2 (13 ng) in EGF-stimulated lysate (40 μg) (Figure S5 in the Supporting Information), which will be an important tool for quantifying ERK1/2 levels in tissue samples.
The MAPK signaling pathways are composed of numerous kinases intricately regulated by stress responses and extracellular signals. Deconvoluting the specific functions of individual enzymes has been challenging, partly due to the difficulty of creating probes that exclusively target a kinase of interest. Here we have presented a selective ERK1/2 activity chemosensor that comprises both a chemical sensing motif and recombinant enzyme docking domain. Thereby, the Sox-based kinase-sensing strategy has been extended beyond the realm of enzymes that recognize linear peptide substrates. Quantitative studies with the probe indicate that the PNT domain confers exquisite selectivity toward ERK1/2, which was impossible to achieve with simple peptide probes. Moreover, the docking-domain approach now allows us to target a wider set of kinases (such as other members of the MAPK family, JNK and p38) that have thus far been elusive due to their complex substrate recognition mechanisms. The chimeric protein probe also offers distinct advantages for solution-based analyses that can be carried out with simple equipment. The reliable semisynthesis of multimilligram (6 mg) quantities of Sox-PNT allows at least 5000 assays to be performed in 384-well plates. Moreover, Sox-based sensors exhibit large dynamic ranges with excellent Z‘ factor values, which are a measure of the fidelity of assay data. Currently, in light of the efficient semisynthesis, excellent selectivity and robustness in high throughput analysis, the Sox-PNT sensor can be broadly applied for quantifying ERK1/2 activities in applications ranging from drug discovery to diagnostics.
**We thank Prof. K. A. Dalby for providing DNA of the PNT domain and the PEA-15 protein, and Dr Kevin Janes for helpful discussions. This research was supported by the NIH Cell Migration Consortium (GM064346). The Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (NSF-0070319) is also gratefully acknowledged.