The tool better honed: Modern cytometric approach to protein phosphorylation

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The Nobel Prize in Physiology or Medicine in 1992 was awarded to Edmond H. Fischer and Edwin G. Krebs for their discovery of reversible protein phosphorylation and its importance as a biological regulatory mechanism. Since their discovery, protein phosphorylation gained most deserved attention of both research-oriented and application-oriented biomedical scientists, as a major process ascertaining and controlling the activity of proteins. The term “signal transduction,” albeit including many different facets of behavior of the involved proteins, conducting the “signal” from the receptor on the cell surface into the nucleus, is sometimes understood as identical with the phosphorylation cascade of a chain of (mostly membrane-bound and cytoplasmic) proteins. It has been long recognized that protein phosphorylation is universal and crucial for the proper initiation and progression of the cell cycle [recently reviewed by Holt (1)], response to hormones and other ligands of the membrane receptors (2), etc. It would be hard to bring up all important facets of the protein phosphorylation process in this short commentary. However, it is not a gross exaggeration to say that the singular process of addition or subtraction of a phosphate group to or from the bulk of a (any) protein (relatively simple from the chemical point of view), is among the most important biochemical process discovered and studied. The knowledge of this process, both in its qualitative (which proteins, at what positions, etc.) and quantitative (e.g., what proportion of the protein of interest gets (de)phosphorylated upon stimulation) aspects bears heavy on our understanding of the processes of life, spanning from the egg fertilization to embryo development to the functioning of any single cell, and finally to cellular ageing and death. Phosphorylation/dephosphorylation processes occurring in specific cell types, notably the cells of the immune system, had been (and still are) intensely studied, both in experimental animals and in human samples. Moreover, it has been discovered over the decades that impaired protein phosphorylation is the culprit of the immune diseases and cancers [reviewed in Ref.3]. Thus, the enzymes adding phosphate groups to proteins—kinases—are already the targets of successful pharmacological interventions in oncology, and they emerge also as targets in the treatment of immune diseases (4).

Let us consider the settings of the immune system, from the point of view of the phosphorylation/dephosphorylation processes. Human genome contains at least 518 kinases in eight groups (the division based mostly on their target specificity and cellular function), of which many, if not the most, are active in various immune cells, performing their respective tasks. Then, of 40+ proteins included in the immune signalosome and involved in the signal transduction from a T(B)-cell receptor to the nucleus (culminating in the transfer of activated transcription factors NFkB, NF-AT, and the like into the nucleoplasm), at least about 20 are kinases and most (if not all) undergo an activating or inhibitory phosphorylation at some or other moment during the first crucial minutes of lymphocyte activation. Quoting the (cited above) paper by Kontzias et al., “The first step in signaling by multi-chain immune recognition receptors, which include the TCR, BCR, Fc receptors and others, is tyrosine phosphorylation of the receptor itself and associated adapter molecules like LAT (linker for activation of T cells). This is mediated initially by Src family protein tyrosine kinases, followed by kinases such as Syk (spleen tyrosine kinase) or Zap-70, Tec family PTKs and later by serine-threonine kinases, such as mitogen-activated protein kinases (MAPKs) and protein kinase C (PKC) family.” And further: “Initial protein phosphorylation ultimately links membrane events to calcium modulation, cytoskeletal rearrangement, gene transcription, and other canonical features of lymphocyte action.” (4). Also the cytokine signaling via the Type I and II cytokine receptors requires the activation of receptor-associated Janus kinases (JAKs), while cytokines belonging to the families of stem cell factor or transforming growth factor bind to receptors possessing respectively the tyrosine or serine-threonine kinase properties. Finally, even if the receptor itself is not a kinase (as is the case for the IL-1 and TNF receptors), the cascades of events initiated by their ligation by respective cytokine includes multiple kinases, i.e. multiple phosphorylation events. Thus, the immune response effectiveness depends heavily on the proper occurrence (timing) and levels of the protein phosphorylation. It is not surprising therefore that the methodology aiming and more and more precise detection, quantification and protein-wise dissection of the process in various cell types and especially the lymphocytes had been developed since its discovery.

Now, the structure of the immune system, being composed of literally scores of cell types (populations and sub-populations), each possessing a unique physiological role in the immune reaction and usually in some way or another participating in them, makes impractical the classic biochemical approach to the detection and quantification of phosphoproteins involved. The first major reason for this impracticality of Western blotting or similar, even the most modern, phosphoproteomic technologies in the study of phosphorylation—involving events in the immune cells' activation is the sheer complexity of the cellular system involved, with multiple direct contact and cytokine—cytokine receptor interactions necessary to occur to ascertain proper level and intensity of the immune reaction; thus, the cells of interest cannot be purified and enriched prior to the study of phosphorylation events. Although such a possibility would exist after the stimulation took place, the proportions of responding cells in case of an antigen stimulation are usually very low, as are the proportions of certain specific subpopulations of interest, e.g. the regulatory T cells (Tregs). Their isolation for the phosphoproteomic/WB platform would require huge amounts of biological material, unavailable especially if human immunity is studied.

Here comes multiparameter cytometry, as the most appropriate current approach to the abovementioned technical problems when attempting the study of phosphorylation processes in the immune system. Some crucial events involving the phosphorylation of signal transduction-associated proteins in the activated T lymphocytes and utilizing the flow cytometric techniques were recently described in Cytometry A (5, 6). The recent paper by Goldeck et al. (7) published in Cytometry A, and coming from the group led by Dr. Anis Larbi in Singapore, is the most recent, comprehensive piece of good scientific work concerning the application of multiparameter flow cytometry to the detection of the phosphoproteins in the mixture of interacting immune cells. As the authors state in the abstract: “Recent advances in immunology and the identification of T lymphocyte sub-populations making up only a very small fraction of the total population highlight the importance of studying signaling in those small subsets in a feasible, cost-effective, high-throughput manner. To this end, we have developed a simplified protocol to study both intracellular phosphorylation patterns of important signal transduction molecules concomitantly with T cell surface marker expression.” And, they deliver as promised. It is a very good technique-describing, but otherwise original paper, most timely and appropriate for the Cytometry readers.

Despite the authors describing their protocol as “simplified,” the method as described allows for simultaneous quantification of as many as five phosphorylated target (signaling) molecules in four major subpopulations of CD4 and CD8 T cells, including naive, central memory, effector memory, and TEMRA. Using the proposed protocol, it is quite easy to obtain the phosphorylation status of at least four to five molecules involved in the T cell activation, namely the Lck, ZAP70, p38, and ERK1/2 simultaneously in at least four abovementioned, different subpopulations of either CD4+ or CD8+ cells. Based on the detailed protocol description and reagent availability, the technique can be easily modified to accommodate actual needs and instrument capabilities in any flow cytometry laboratory. It is clearly “downgradeable”—the researcher does not have to use the “top shelf,” -teen parameters' flow cytometer if the experiment design does not ask for all the parameters described in the paper—but can also be further developed (upgraded) for those even more multi-parameter studies feasible and predictable in the future, with the development of both cytometric hardware as well as the corresponding, powerful new software.

The method allows for obtaining meaningful results from as few as 1 × 106 cells per assay. As the authors say, “Minimizing the method further was possible (0.5 × 106 PBMC) but within limits, because some T cell subsets are in present at low abundance” which obviously puts some limits on the technique, but is not its intrinsic fault. Careful to the details, the paper points our attention to the apparently trivial elements of the protocol, like the choice of fixation-permeabilization reagents and of the antibody-fluorochrome conjugates, showing that these are not trivial after all.

Another interesting observation (albeit treated a bit marginally in the paper, as not being its major aim) is the demonstration of difference in phosphorylation levels between the lymphocytes treated identically with one exception—culturing in the hypoxic atmosphere of 2% oxygen and 5% carbon dioxide in nitrogen versus typically used 5% carbon monoxide in air atmosphere. The readers should be on a look-out for more such papers comparing the behavior of immune cells under standard conditions and under those hypoxic (but apparently more close to physiological) ones.

The authors are modest enough to say “Our protocol is one amongst several”; however, the search of PUBMED™ database does not yield many such “technical” papers. A few recent and comparable works have been recently published (8, 9). Of course it is up to the individual researchers to decide which of the techniques to apply to their research. Still, concluding, I believe this paper to be the “must read” for anybody interested in or currently studying the protein phosphorylation events in the setup of human or animal immune systems.

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