Laurie L. Parker

Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine.

Background: Cdc14 phosphatases help control mitosis by dephosphorylating sites (Ser/Thr-Pro) targeted by cyclin-dependent kinases (Cdks). Results: Cdc14 family phosphatases strongly prefer phosphoserine over phosphothreonine at Cdk sites. Conclusion: By discriminating among Cdk sites, Cdc14 may participate in setting the order and timing of Cdk substrate dephosphorylation. Significance: Mechanisms governing the timing of Cdk site dephosphorylation are crucial for proper coordination of late mitotic events. Mitotic cell division is controlled by cyclin-dependent kinases (Cdks), which phosphorylate hundreds of protein substrates responsible for executing the division program. Cdk inactivation and reversal of Cdk-catalyzed phosphorylation are universal requirements for completing and exiting mitosis and resetting the cell cycle machinery. Mechanisms that define the timing and order of Cdk substrate dephosphorylation remain poorly understood. Cdc14 phosphatases have been implicated in Cdk inactivation and are thought to be generally specific for Cdk-type phosphorylation sites. We show that budding yeast Cdc14 possesses a strong and unusual preference for phosphoserine over phosphothreonine at Pro-directed sites in vitro. Using serine to threonine substitutions in the Cdk consensus sites of the Cdc14 substrate Acm1, we demonstrate that phosphoserine specificity exists in vivo. Furthermore, it appears to be a conserved property of all Cdc14 family phosphatases. An invariant active site residue was identified that sterically restricts phosphothreonine binding and is largely responsible for phosphoserine selectivity. Optimal Cdc14 substrates also possessed a basic residue at the +3 position relative to the phosphoserine, whereas substrates lacking this basic residue were not effectively hydrolyzed. The intrinsic selectivity of Cdc14 may help establish the order of Cdk substrate dephosphorylation during mitotic exit and contribute to roles in other cellular processes.

Time-Resolved Luminescence Detection of Spleen Tyrosine Kinase Activity through Terbium Sensitization

Disruption of regulatory protein phosphorylation can lead to disease and is particularly prevalent in cancers. Inhibitors that target deregulated kinases are therefore a major focus of chemotherapeutic development. Achieving sensitivity and specificity in high-throughput compatible kinase assays is key to successful inhibitor development. Here, we describe the application of time-resolved luminescence detection to the direct sensing of spleen tyrosine kinase (Syk) activity and inhibition using a novel peptide substrate. Chelation and luminescence sensitization of Tb(3+) allowed the direct detection of peptide phosphorylation without any antibodies or other labeling reagents. Characterizing the Tb(3+) coordination properties of the phosphorylated vs unphosphorylated form of the peptide revealed that an inner-sphere water was displaced upon phosphorylation, which likely was responsible for both enhancing the luminescence intensity and also extending the lifetime, which enabled gating of the luminescence signal to improve the dynamic range. Furthermore, a shift in the optimal absorbance maximum for excitation was observed, from 275 nm (for the unphosphorylated tyrosine peptide) to 266 nm (for the phosphorylated tyrosine peptide). Accordingly, time-resolved measurements with excitation at 266 nm via a monochromator enabled a 16-fold improvement in base signal-to-noise for distinguishing phosphopeptide from unphosphorylated peptide. This led to a high degree of sensitivity and quantitative reproducibility, demonstrating the amenability of this method to both research laboratory and high-throughput applications.

Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

Many cancers exhibit deregulated activity of protein kinase enzymes, but not all are sensitive to inhibitor drugs, largely because phosphorylation dynamics in complex tissues are not well understood.[1] Live, subcellular analysis can reveal the details of kinase signaling in mixed populations of cells.[2] Current tools to image kinase activity in situ depend on intensity-based measurements (such as fluorescence and Forster resonance energy transfer) that can be limited by spectral bleed-through and photobleaching.[3] We report a cell-penetrating peptide biosensor for dynamic monitoring of phosphorylation by Abl kinase based on fluorescence lifetime imaging microscopy (FLIM).[4] FLIM, which is not confounded by photobleaching or cellular autofluorescence, was applied to detect phosphorylation-dependent fluorophore lifetime shifts (1–2 ns) in intact, living cells (Fig. 1). We established the dependence of the fluorophore lifetime shift on phosphorylation specifically by Abl kinase, mapped the fluorophore intensity and lifetime components to quantify subcellular phosphorylation, and monitored kinase inhibition in real time. This approach should be generalizable to other kinases and provides a new method for interrogating real-time, subcellular signaling activities in cell populations that are not amenable to expression of genetically engineered biosensor proteins. Figure 1 FLIM to detect phosphorylation-dependent fluorophore lifetime shifts for biosensors in intact, live cells Measuring subcellular kinase activity in living cells remains a major challenge. Genetically encoded Forster resonance energy transfer (FRET) biosensors can be used for this purpose in simple cell-based assays and basic research applications.[3, 5] These sensors take advantage of binding between phosphorylated sequences and phosphopeptide binding domains to bring two fluorescent proteins close enough for energy transfer to occur. However, expressing genetically engineered proteins in cells has challenges, including a) uniform transfection and expression of protein fluorophores (a roadblock for applications in primary patient-derived cells or tissues) and b) the large labels which can affect substrate function and interaction with a kinase.[5–6] Small molecule fluorophores are able in principle to be less disruptive to function, and many are available for which excitation and emission do not overlap with expressible fluorophores (enabling multiplexed co-localization experiments).[7] These have been applied to detect phosphorylation in cells via fluorescence intensity increases.[8] Low signal to noise is a limitation of FRET, and intensity-based fluorescence is confounded by photobleaching when experiments are conducted over long time periods, making it difficult to interpret subcellular fluctuations at high spatial and temporal resolution.[6] FLIM is not affected by photobleaching or intensity and has the potential for single molecule monitoring.[4, 9] Also, time-correlated single photon counting FLIM is capable of highly resolved discrimination between species exhibiting very small differences in lifetimes (even sub-nanosecond), facilitating the mapping of exquisite detail in subcellular images. Here we describe the first demonstration of a FLIM-based phosphorylation biosensor technology that has the potential to circumvent key technological gaps as a new strategy for studying intracellular signaling biology. We combined the delivery of organic fluorophore-tagged kinase substrate peptide probes with time-resolved FLIM to visualize the details of kinase activity in live, intact cells (Fig. 1). The biosensor consists of an Abl substrate peptide containing the “Abltide” substrate sequence[10] tagged with a Cy5 fluorophore and a cell penetrating peptide (Abl-TAT: GGEAIYAAPCCy5GGRKKRRQRRRPQ) (Fig. 2).[11] The substrate portion (bold) is relatively selective for the c-Abl kinase (Abl1) over other tyrosine kinases, however it is phosphorylated by the Abl family member named Abl-related gene (Arg, also known as Abl2).[12] Abl1 and Abl2 are highly homologous and share many functions in normal cells.[13] In this work, “Abl kinase” denotes both Abl1 and Abl2. We used FLIM instrumentation with picosecond pulsing lasers[9a, 9b] to measure Cy5 lifetime for the unphosphorylated biosensor and a phosphorylated derivative in solution and in live cells. Figure 2 Peptide-based Abl kinase biosensor In solution, lifetime differences between the phosphorylated and unphosphorylated Abl-TAT peptide species were not significant (see supporting information, Fig. S1a), indicating that phosphorylation alone is not sufficient to elicit a change in the rate of fluorescence signal decay for the Abl-TAT peptide sensor. However, in the presence of c-Abl kinase at 1:1 ratio, robust lifetime differences were observed, and this phenomenon was blocked by pre-incubation of the kinase with higher ratios of unlabelled phosphopeptide (Fig. S1b). This effect likely arises from the more drastic change in the local environment of the fluorophore that could occur upon binding of the phosphopeptide with the protein, probably through the kinase SH2 domain.[14] Since the physiochemical basis for the lifetime shift was still somewhat unknown, standards were established in NIH3T3 immortalized mouse embryonic fibroblast cells (MEFs)[15] to assess phosphorylation- and Abl kinase-dependence of the lifetime shifts by using three key negative controls (which exhibited lower lifetimes in cells): Cy5 alone, a non-phosphorylatable Y→F peptide sensor analog (Abl-F-mutant) (both in control MEFs expressing Abl kinase) and the Abl-TAT biosensor in Abl(−/−) knockout cells[15] (Fig. 3B, C and E and Fig. S2). Average lifetimes per cell for multiple cells were calculated and plotted to show the distribution of lifetimes observed for the biosensor and each control (Fig. 3G). The distributions were determined to be non-Gaussian, so non-parametric ANOVA with a Dunn’s post-test (described in the Methods section) was used to evaluate statistical significance (P<0.05) for differences in the mean lifetimes. There was no significant difference between the Cy5, Abl(−/−) or Abl-F-mutant experiments, however each of these controls exhibited significantly lower mean lifetimes than both Abl-TAT (in MEFs expressing Abl kinase) and Abl-phospho (positive control). The mean lifetime for Abl-phospho was also significantly longer than that of Abl-TAT, consistent with enrichment of the phosphorylated form of the substrate. These experiments confirmed that the increase in Cy5 lifetime was specific and due to Abl dependent phosphorylation of the biosensor peptide on tyrosine. As another control to support the interpretation of phosphorylation dependence for lifetime increases in cells, we used immunocytochemistry to show colocalization between the Abl-TAT biosensor and phosphotyrosine (see supporting information, Fig. S3). Figure 3 FLIM mapping of biosensor phosphorylation We tested sequestration of the biosensor in endosomes by staining for an endosomal marker. Minimal, non-exclusive colocalization of the biosensor with endosomes was observed, indicating that the biosensor peptide was not sequestered (Fig. S3). We also examined peptide degradation, a potential issue in some cell types[16] but not all, as demonstrated from our prior work.[11a] Controls using fluorescence correlation spectroscopy (FCS) measured Cy5 alone vs. the Abl-TAT biosensor in MEFs (supporting information, Figure S4), providing evidence that for MEFs, the signal observed for the Abl-TAT biosensor arose from peptide that was not degraded to free Cy5.[17] To quantitatively address the distribution and level of biosensor phosphorylation in different subcellular regions we separated the intensity and lifetime components of the signal arising from the FLIM measurements and plotted lifetime values in 2D using MatLab (as shown in Fig. 3A–F). We did not observe phosphorylation-dependent intensity increases for this biosensor either in solution or in cells (Fig. S2A) (in contrast to what has been observed by others).[8a, 18] We then used the Abl-TAT biosensor to image Abl kinase inhibition with the kinase inhibitor imatinib in control MEFs (Fig. 4). MEFs stably expressing a nuclear-enriched Abl kinase mutant, FKBP-Abl(NUK),[15] were also analysed in the presence of imatinib (see time lapse movie shown in Fig. S5). Over the course of 70 min we detected a general trend towards decreased lifetime overall (e.g. Fig. 4 and Fig. S5) and negative lifetime shifts in multiple areas of the cell within the first few minutes of incubation (Fig. S6), consistent with kinase inhibition and dephosphorylation of the biosensor by phosphatase enzymes (previously observed[11a]). In the absence of imatinib, biosensor lifetime was dynamic but overall no significant decrease in lifetime was observed (Fig. 4). Figure 4 Subcellular Abl inhibition by imatinib These experiments demonstrate that fluorescence lifetime shifts measured for the cell-deliverable kinase biosensor are phosphorylation-dependent, yielding dynamic information about the localization of kinase (and potentially phosphatase) activity in single living cells. This could make it possible to examine heterogeneous mixtures of cells to dissect subsets of signaling phenotypes and responses to inhibitors. This approach should also be generalizable to other kinase substrates and fluorophores, enabling the future possibility of analyzing more than one kinase-targeted FLIM biosensor at a time. Currently, we are developing other kinase substrate biosensors (e.g. for the Syk kinase[19]) to expand the application of this strategy and achieve simultaneous detection of multiple kinase activities in situ.

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates

Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.

KINATEST-ID: A Pipeline To Develop Phosphorylation-Dependent Terbium Sensitizing Kinase Assays

Nonreceptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb(3+)-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align, and select potential phosphorylation-dependent Tb(3+)-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to high-throughput screening (HTS) applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use HTS-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein-binding ligands.

A Peptide-Based Biosensor Assay To Detect Intracellular Syk Kinase Activation and Inhibition

Spleen tyrosine kinase (Syk) has been implicated in a number of pathologies including cancer and rheumatoid arthritis and thus has been pursued as a novel therapeutic target. Because of the complex relationship between Syks auto- and other internal phosphorylation sites, scaffolding proteins, enzymatic activation state and sites of phosphorylation on its known substrates, the role of Syks activity in these diseases has not been completely clear. To approach such analyses, we developed a Syk-specific artificial peptide biosensor (SAStide) to use in a cell-based assay for direct detection of intracellular Syk activity and inhibition in response to physiologically relevant stimuli in both laboratory cell lines and primary splenic B cells. This peptide contains a sequence derived from known Syk substrate preference motifs linked to a cell permeable peptide, resulting in a biosensor that is phosphorylated in live cells in a Syk-dependent manner, thus serving as a reporter of Syk catalytic activity in intact cells. Because the assay is compatible with live, primary cells and can report pharmacodynamics for drug action on an intended target, this methodology could be used to facilitate a better understanding of Syks function and the effect of its inhibition in disease.

Detection of early Abl kinase activation after ionizing radiation by using a peptide biosensor.

The ubiquitously expressed Abl protein is a non‐receptor tyrosine kinase that undergoes nuclear–cytoplasmic shuttling and is involved in many signaling pathways in the cell. Nuclear Abl is activated by DNA damage to regulate DNA repair, cell‐cycle checkpoints and apoptosis. Previous studies have established that ataxia telangiectasia mutated (ATM) activates nuclear Abl by phosphorylating serine 465 (S465) in the kinase domain in response to ionizing radiation (IR). Using a peptide biosensor that specifically reports on the Abl kinase activity, we found that an Abl‐S465A mutant, which is not capable of being activated by ATM through the canonical site, was still activated rapidly after IR. We established that DNA‐dependent protein kinase (DNAPK) is likely to be responsible for a second pathway to activate Abl early on in the response to IR through phosphorylation at a site other than S465. Our findings show that nuclear and cytoplasmic Abl kinase is activated early on (within 5 min) in response to IR by both ATM and DNAPK, and that although one or the other of these kinases is required, either one is sufficient to activate Abl. These results support the concept of early Abl recruitment by both the ATM and the DNAPK pathways to regulate nuclear events triggered by DNA damage and potentially communicate them to proteins in the cytoplasm.

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

The majority of eukaryotic proteins are phosphorylated in vivo, and phosphorylation may be the most common regulatory posttranslational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases and can be applied to studies of other protein modifications as well.

Multicolored, Tb3+-Based Antibody-Free Detection of Multiple Tyrosine Kinase Activities

Kinase signaling is a major mechanism driving many cancers. While many inhibitors have been developed and are employed in the clinic, resistance due to crosstalk and pathway reprogramming is an emerging problem. High-throughput assays to detect multiple pathway kinases simultaneously could better model these complex relationships and enable drug development to combat this type of resistance. We developed a strategy to take advantage of time-resolved luminescence of Tb(3+)-chelated phosphotyrosine-containing peptides, which facilitated efficient energy transfer to small molecule fluorophores conjugated to the peptides to produce orthogonally colored biosensors for two different kinases. This enabled multiplexed detection with high signal-to-noise in a high-throughput-compatible format. This proof-of-concept study provides a platform that could be applied to other lanthanide metal and fluorophore combinations to achieve even greater multiplexing without the need for phosphospecific antibodies.

A Multiple Reaction Monitoring (MRM) Method to Detect Bcr-Abl Kinase Activity in CML Using a Peptide Biosensor

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.