Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity
© Green and Alberola-Ila; licensee BioMed Central Ltd. 2005
Received: 05 January 2005
Accepted: 05 July 2005
Published: 05 July 2005
Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity.
ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK.
EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.
Traditional methods for studying signal transduction cascades have been based solely on biochemical analysis of whole cell populations and homogenized tissues (e.g. radioassays, western blots, etc.). In addition, in vitro studies have required the use of radioactive isotopes for biochemical characterization of kinases. These methods are time consuming, produce large quantities of radioactive waste, and do not allow for the study of real-time kinase dynamics.
Recently, sensors for studying signal molecules in vitro, as well as cascade dynamics in single cells, have been developed utilizing fluorescent proteins and the phenomenon of fluorescence resonance energy transfer (FRET). FRET is a phenomenon by which energy is transferred from one fluorescent molecule to another by way of dipole-dipole interactions during excitation of the donor molecule. FRET efficiency is given by the equation:
where R is the donor-acceptor radius and Ro is the radius at which FRET efficiency is 50% (Förster radius). Small changes in R (1–2Å) and small orientation changes between the donor and acceptor fluorophores can dramatically affect the efficiency of FRET, making very small changes in structure easily detectable. The limitation, however, is that FRET is effective only between 10–100Å, and therefore the donor and acceptor must be maintained in close proximity. Genetically encoded fluorescent proteins fused by linker peptide sequences have been utilized to address proximity limitations, as well as facilitate the delivery of the sensor into live cells.
Some of the earliest work done with these sensors includes genetically encoded sensors of caspase activity, in which caspase-3 and caspase-8 sensitive linker peptides were fused between the different variants of Enhanced Green Fluorescent Protein (EGFP) . These studies allowed the characterization of apoptosis in single living cells with spatio-temporal resolution. Other types of FRET signaling sensors have since evolved, including sensors for calcium signaling , receptor tyrosine kinases [3, 4], intracellular kinases [4–7], and histone methylation . These sensors have been shown to be useful in in vitro assays, as well as allow the study of signaling events in a single cell, in real time .
The Ras/ERK cascade controls differentiation and proliferation in many different cell types and organisms. In this signal transduction pathway, activated Ras (Ras-GTP) binds directly to Raf-1 and recruits it to the membrane where Raf becomes activated. Raf then phosphorylates and activates Mek-1 and Mek-2 (the MAPK kinase), which in turn phosphorylate the MAPKs ERK-1 and ERK-2. Activated ERKs translocate to the nucleus and directly phosphorylate transcriptional regulatory proteins (including members of the Ets family of transcription factors, and the bZIP factors Fos and Jun) (reviewed in ). Kinetics and in vivo dynamics of ERK MAP kinase activity are not completely understood. Traditionally, the level of ERK activation has been thought to be relative to the strength of the upstream signal. Some data suggest, however, that ERK activation is "switch-like," requiring a threshold of activation, above which all ERK molecules within the cell become activated . Computational models of MAPK signaling mechanisms can fit both possibilities [11, 12]. Therefore, the development of new tools to study these dynamics is essential to determine the biochemical nature of ERK signaling.
In this manuscript, we describe the development of several FRET-based sensors of MAP kinase activity which we have called ERK Activity Sensors (EAS). Peptide linker sequences taken from the Ets family transcription family members Ets1 and Elk1, both ERK targets, were fused between cyan and yellow variants of EGFP (ECFP and EYFP, respectively). Several of our constructs respond to phosphorylation by activated ERK with changes in FRET, and at least one of them, EAS-3, is specific for active Extracellular Regulated Kinase (ERK) as opposed to two other MAP kinases, p38 and SAPK/JNK. Therefore, EAS-3 is a viable, non-radioactive sensor of ERK MAPK activity in vitro.
Results and discussion
Design and model of EAS
EAS FRET changes in the presence of active ERK2
EAS changes in FRET in response to pERK2 require phosphorylation and the ERK binding site
Mutants of EAS-3 were utilized to validate the structural features of EAS essential for decreased FRET efficiency upon incubation with pERK2. We refined EAS-3 by replacing the bulky N-terminal GST-purification tag (Glutathione-S-Transferase) with a Histidine-10 tag on the C-terminus. This reduced the possibility that the large purification tag would interfere with FRET efficiency changes. In addition, we mutated alanine 207 and alanine 487 to lysine. As previously reported, this prevents EGFP dimerization . This latter modification reduced co-purification of truncation products with full-length EAS (data not shown).
EAS-3 is not phosphorylated by pSAPK or pp38
Distinguishing the activation of different MAP kinases within the cell is essential since each MAPK pathway is activated by multiple mitogens and external environmental factors to varying degrees (reviewed in ). Furthermore, there is extensive cross-talk between the different MAP kinase pathways. Detection methods must effectively isolate the signal of the target kinase from other family members to elucidate the contributions of these different pathways to a given cellular process.
The demonstrated specificity of pERK2 for EAS-3 phosphorylation suggests that this sensor is a candidate for in vivo studies of ERK signaling. However, our preliminary experiments in NIH-3T3 cells indicated that EAS-3 was susceptible to intracellular phosphatase activity (data not shown). We surmise that this is due to the absence of a protective phospho-specific binding domain within the EAS-3 construct. Such binding domains have been crucial in the development of other FRET-based signaling sensors of kinase activity [2–4, 6, 7]. These binding domains, however, are taken from naturally occurring domains that are specific for each target sequence. Unfortunately, no known phospho-specific binding domain exists for Elk1 in the region of Ser383/389. Therefore, we are using a semi-rational approach to develop a phospho-specific binding domain that acts to protect the phosphorylated EAS-3 linker from intracellular phosphatases. This will enable us to adapt the EAS-3 sensor for use in live cells.
These results show that our novel ERK Activity Sensors provide real-time in vitro detection of MAP kinase activity. This method can be applied to studying kinetics of ERK activity in real time, as well as detection of ERK activity in unknown cell lysate fractions. There is also the potential to use these sensors for high-throughput screening of ERK kinase activity with fluorescence plate readers. This method is more direct and convenient to monitor ERK activation in vitro than conventional assays that either use radioactivity for detection or rely on indirect detection using phospho-specific antibodies for MAPK targets.
DNA coding for peptide linkers was amplified by PCR with primers designed with Bgl II and BamHI sites at the 5' and 3' ends, respectively. The products were cloned into pECFP-C1 (Clontech), and EYFP from pEYFP-N1 was subsequently cloned in frame to create EAS constructs. EAS's were cloned into pGEX-2T (Amersham) in frame with GST for expression and purification purposes. These GST-tagged constructs were used for initial fluorimetry experiment of EAS constructs. Other versions of EAS's were constructed using ECFP and EYFP with mutation A(207,487)K, which eliminates dimerization of GFPs, and cloned into pET21a (Novagen) with a Histidine-10 tag. These His10-tagged constructs were used for mutant and specificity experiments. EAS-3 linker mutants were made by Quickchange Site Directed Mutagenesis (Stratagene) to make either single or double point mutation within the peptide linker. For expression in NIH-3T3 cells, EAS's were cloned into the pECFP vector backbone without tags.
EAS and active kinase expression
BL21(DE3) cells were transformed with EAS constructs and plated on LB agar supplemented with 100 μg/ml ampicillin. Colonies were picked into 4 ml LB-Amp and shaken overnight at 30°C. 250 ml LB was inoculated with starter culture, grown to A600 of 0.6, and induced with 0.1 mM IPTG for 18 hours. Cells were spun down, lysed by french press, and cell fragments were spun down at 18,000 rpm in a Beckman JA-20 rotor for 30 min. Proteins were purified from lysates over Ni-NTA Superflow (Qiagen) or Glutathione Sepharose 4B (Amersham).
Activated kinases were purified as described . pERK2 was either purchased (NEB) or purified from bacteria transformed with a plasmid containing both constitutively active MEK1* and His-tagged ERK2. Phosphorylation of ERK2 by MEK1* occurred in bacteria prior to lysis. BL21(DE3) cells were electroporated with MEK1*/ERK2 construct and grown overnight at 37°C on LB-Amp agar plates. Several colonies were picked and incubated in 4 ml of TB supplemented with 100 μg/ml carbenicillin. The starter culture was added to 1L of TB, grown to A600 of 0.35, and then induced with 0.25 mM IPTG for 12 hours at 30°C. Cells were harvested and lysed as above, and purified over Ni-NTA Superflow. Activated p38 and SAPK/JNK were purified from bacteria electroporated with two plasmids, one coding for constitutively active MEK kinase 4 (MEKK4*) and another coding for MEK4 and either His-tagged p38 or His-tagged SAPK/JNK. These constructs were grown and purified as above, except with both carbenicillin and kanamycin (50 μg/ml). Protein concentration and buffer exchange performed with Centriplus, Centricon, and Microcon ultrafiltration membranes (Millipore).
Kinase assays and fluorimetry
Radioactive kinase assays were performed in 25 μl in 12.5 mM MOPS pH 7.5, 12.5 mM β-glycerophosphate pH 7.3, 7.5 mM MgCl2, 500 μM NaOrthovanadate, 500 μM NaF, and 9.7 nM DTT. Amounts of kinase and substrate added to reactions are indicated in figure legends. Finally, ATP supplemented with 0.2 nmol [γ-32P]ATP (6000 mCi/mmol, Molecular Bioproducts), was added to a final concentration of 1 mM. Reactions were incubated at 30°C for 15 minutes, 8.3 μl of 4× protein sample buffer (SB) were added, and samples were boiled for 5 minutes. Samples were analyzed with 10% or 12% SDS-PAGE (BioRad Mini-Protean II) and transferred to a nitrocellulose membrane. The membrane exposed to a phosphoscreen and scanned on a Storm 860 (Molecular Dynamics). Quantitative densitometry of bands was performed using ImageQuant 5.0 (Molecular Dynamics).
Fluorimetry performed in a Shimadzu Spectrofluorophotometer RF-5301PC. Assays were performed in same buffer as above in a volume of 2.5 ml. The final concentration of EAS constructs was 250 nM and final concentration of pERK2 was 50 nM. The reaction was incubated in fluorimeter and warmed to 30°C. Excitation was set at 425 nm to excite ECFP and avoid excitation of EYFP and readings were taken in 0.2 nm increments. An initial reading was taken prior to ATP addition. To initiate reaction, ATP was added to a final concentration of 1 μM, a spinner was activated for 1 minute, and first reading taken at 2 minutes. EYFP to ECFP ratios were calculated by dividing EYFP peak emission by ECFP peak emission. Peak emissions were defined as an average intensity of EYFP between 524.6–525.4 nm and of ECFP between 474.6–475.4 nm.
We would like to thank Dr. Kornfeld for providing us with GST-Elk1 cDNA and Dr. Sharrocks for providing us with Ets1 cDNA, from which EAS linkers were derived. We also thank Dr. Richard Roberts for use of his fluorimetry equipment. This work was supported by the NIH (AI45072) and the Keck Foundation.
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