Fluorogenic Cell-Based Assays
Wen-Chieh Liao, research scientist at R&D Systems described a fluorogenic cell-based ELISA that does not require lysate preparation or the multiple steps required to achieve Western blot protocols for analysis of cellular proteins and phosphorylation. This method is amenable to high-throughput applications and may, Dr. Liao said, prove a valuable addition to kinase inhibitor screening strategies.
The relatively rapid and straightforward method, according to Dr. Liao, provides a way to analyze multiple samples and enables analysis of two target cellular proteins or events simultaneously in the same microplate wells to minimize assay variability. Dr. Liao said these cell-based ELISAs have been used to evaluate the effects of stimulators and inhibitors on cultured cells with 10,000 cells, or fewer, per well.
He described phosphorylation of JNK (T183/Y185), Akt (S473), EGFR (Y1068), and TAT3 (Y705) in response to various stimuli or kinase inhibitors. In contrast to other techniques for intracellular protein analysis, including Western blots and sandwich ELISA, Dr. Liao said, total hands-on time for the cell-based ELISA amounts to about three hours, while conventional assays require about two days because of the need to prepare lysates and other laborious steps.
In describing what specific issues with conventional assays this technology addresses, Dr. Liao explained that “cell-based assays are associated with poor accuracy and reproducibility due to well-to-well variations such as cell numbers. However, our assays simultaneously measure two proteins in one well, thus allowing for normalization of well-to-well variations.”
Analyzing phosphoproteins by Western blot requires preparation of cell lysates and does not allow the study of many samples that are essential for high-throughput drug screenings, he said.
“In contrast, our dual-fluorescence cell-based ELISA allows researchers to grow their cells in 96-well plates, treat them with appropriate conditions, and then quickly fix and permeabilize in the wells. This is followed by incubation of cells with two primary antibodies against proteins of interest. Then, two secondary antibodies labeled with HRP and AP, respectively, and spectrally distinct fluorogenic substrates for each enzyme are used for detection.
“In this assay format, two target cellular proteins or events can be analyzed simultaneously in two fluorescence channels, thus allowing users to improve accuracy by normalizing for well-to-well variations.” Since the assay detection allows for measurement with a standard fluorescence plate reader, there is no need for specialized and expensive equipment.
Although in vitro biochemical kinase assays are routinely used for drug screening, he said, they cannot replicate the intracellular environment. “Our assay allows drug screening of intact cells, and may reflect physiological conditions more closely.” Dr. Liao and his group have used this assay to evaluate phosphorylation of multiple signaling proteins in response to kinase inhibitors, and to determine the IC50 of such inhibitors such as EGFR, ERK1/2, and PI3-kinase inhibitors.
How cells sort out and balance the complex array of signaling events that occur via phosphorylation and dephosphorylation is a prime focus in the laboratory of Alexandra Newton, Ph.D., professor of pharmacology at the University of California, San Diego.
In her presentation, Dr. Newton discussed the role of phosphatase enzymes in controlling protein kinase activation and deactivation. Dr. Newton’s laboratory specifically focuses on the molecular mechanisms of signal propagation and termination in the PI3 kinase and diacylglycerol signaling pathways, and particularly on how protein kinase C (PKC) and Akt (protein kinase B) are activated by phosphorylation events, by second messengers, and inactivated by dephosphorylation via phosphatases.
For example, Akt is a serine/threonine kinase that is the downstream target of PI3K signaling. This kinase mediates most of the PI3K-mediated metabolic actions of insulin through the phosphorylation of several substrates including other kinases, signaling proteins, and transcription factors.
In addition, its activation plays a key role in cell survival. Misregulation of the balance between the phosphorylation and dephosphorylation events leads to pathogenic states, Dr. Newton said. Notably, tipping the balance toward unregulated activation of Akt is a major contributor to cancer.
In her presentation, Dr. Newton focused on the importance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) in acting as a “brake” to terminate signaling by PKC and Akt.
These Ser/Thr specific phosphatases, discovered in Dr. Newton’s laboratory, have emerged as important regulators of intracellular signaling pathways by specifically dephosphorylating and inactivating the two pro-survival kinases Akt and PKC. By examining phosphorylation of these two kinases following manipulation of PHLPP levels through genetic depletion by siRNA, Dr. Newton and her colleagues were able to show that PHLPP controls PKC levels and its activity in the cell.
Because PHLPP is lost in a large number of cancers, thereby conferring a survival advantage to these cells, PHLPP activators may prove useful as cancer therapeutics. Conversely, PHLLP inhibitors would be very useful for diseases where increased Akt activity would be beneficial, such as heart disease and cancer. Dr. Newton further explained that the catalytic domain of this phosphatase belongs to the PP2C family of serine/threonine phosphatases, for which no specific inhibitors exist.
Following medium-throughput chemical screening combined with a virtual approach, her laboratory identified several small molecule PHLPP inhibitors. Treatment of cells with these compounds, she said, caused an increase in both basal and agonist-evoked phosphorylation and activity. These newly discovered inhibitors of PHLPP may be therapeutically relevant in the treatment of diabetes or cardiovascular diseases, in which Akt and PKC pathways are repressed.
Presentations at this meeting confirmed the tremendous progress that has been made in analyzing cell signaling, and pointed out potential intervention points for the development of novel therapeutics.