New Assay Development: Phospho-STAT5 (pSTAT5)ASSAY
Introduction:
Protein phosphorylation is an important pathway for regulation of protein function in living cells. It is relevant for the process of signal transduction that regulates gene expression, cellular proliferation and differentiation. Cells respond to the environment in a stepwise mechanism. Upon engagement of a receptor (e.g., growth-, hormone-, cytokine-receptor), molecules are recruited to the receptor to relay the signal to the interior of the cell, resulting in a series of events in response to the outside stimulus. The predominant mechanism by which signal transduction occurs is through tyrosine phosphorylation of intracellular proteins, which in turn induces conformational changes in the proteins, allowing other proteins and molecules to dock and interact with the phosphorylated protein. In the case of signal transduction pathways, phosphorylation is often a transient, reversible, process, indicative of the (sequential) activation of proteins within a cascade of transduction events. Thus, measurements of phosphorylation of specific intracellular (intranuclear) proteins provides valuable information regarding the specific pathways that have been engaged, including the origin of the cellular activation as well as the kinetics of the response to the outside stimulus. It has been well recognized that defective phosphorylation is linked to a variety of pathological states, including cancer, asthma, diabetes, inflammatory and immunodeficiency disorders.
Consequently, recognition of this central importance of protein kinase dysfunction has spurred development of specific kinase inhibitors to target certain diseases (e.g. imatinib mesylate (Gleevec) as an inhibitor of the Bcr-Abl signaling protein in chronic myeloid leukemia). Thus comparison of differences in phosphorylation events between healthy and diseased cells could be used to identify aberrant intracellular behavior that underlies certain disorders, both from standpoint of diagnostics and therapeutics. Thanks to advances in the field of flow cytometry (FCM), multi-parameter phospho- FCM is now available to characterize multiple phosphorylated molecules simultaneously at the single cell level, using cells from all relevant tissues (see references 1-4). An increasing number of phosphospecific antibodies recognizing phosphoepitopes (e.g. phospho-tyrosine and -serine) are now available, as are their nonphosphorylated counterparts. These include proteins belonging to the mitogen-activated protein (MAP) cascades, the Janus kinase/ Signal transducer and activator of transcription (Jak/Stat) cascades, and tyrosine kinase receptors. In addition to new FCM reagents, other innovative developments include improved methods for permeabilization, allowing the reagents to bind to the phosphoproteins, new approaches to visualization and analysis of data (akin those used in proteomics), and robotics platforms to facilitate high-throughput acquisition of data.
Jak/Stat Pathway
The Jak/Stat pathways are especially relevant in the study of immunodeficiency and inflammatory disorders. A multitude of cytokines and growth factors converge in an hour-glass fashion, using limited combinations of Jaks and Stats to transduce the signals down-stream and elicit a multitude of immune/inflammatory responses. Simplified, binding of the cytokine/growth factor to the receptor triggers activation of a specific Jak, which subsequently phosphorylates tyrosine residues on the receptor (see figure above). This creates sites for interaction with matching Stat proteins that are recruited to the receptors. In turn, the Stats are tyrosinephosphorylated by the Jaks. These phosphotyrosines then act as docking sites for other Stats, mediating their dimerization into hetero– and homo-dimers. The dimmers accumulate in the cell nucleus and activate transcription of their target genes (in an hour-glass fashion).
Detection of Stat phosphorylation by phospho- FCM can test the integrity of the Jak/ Stat-dependent pathways in a variety of inflammatory and immunodeficiency disorders. Some of these disorders lend themselves for relatively straightforward screening by phospho-FCM of relevant members of the Jak/Stat cascades (see reference 5). Phospho-FCM can test the transduction pathway used by the common gamma chain family of cytokines in lymphocytes. Cell surface cytokine receptors of this family include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-12, while Jak3 and Stat5 represent the convergence of the signal transduction pathway.
The lethal immunodeficiency disorder; Xlinked severe combined immunodeficiency (X-SCID) is caused by mutations in the gene (located on the X chromosome) that encodes the common gamma chain (GC) subunit shared by these cytokine receptors. Boys who have a mutation in this gene, lack T cells and NK cells and have defective B-cell function (despite the presence of B cells). These defects are linked to defective function of multiple cytokine receptors. In addition to X-SCID, a similar autosomal recessive form of SCID has been identified by linking the GC cytokine receptor family to abnormal cellular responses, mediated by Jak3. Since both XSCID and Jak3-mutant SCID converge at the level of Stat5, phospho- FCM of Stat5 would provide a screening tool for these conditions. This principle is represented by the phospho-Stat5 (pSTAT5) assay. In this assay, whole blood is transiently stimulated by IL-2 to activate the pathway. This is followed by detectionof pStat5 in selected lymphocyte populations by a phospho-specific antibody. In the figure below, an example is shown in a healthy adult. The red line represents pSTAT5 expression in the unstimulated condition; the black line represent pSTAT5 expression in CD4+ T cells following stimulation (60% positive). In addition to screening for SCID, the pSTAT assay can also be used to test in vitro B-cell function, as well as measure (constitutive) stat5 phosphorylation in other cells and conditions, such as mast cells, and malignancies. More about this assay and other phospho-FCM applications in future Newsletters.
| Phospho-FCM/pSTAT5; further reading: |
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- Schulz et al. Curr Protoc Immunol. 2007:Unit 8.17
- Nolan. Hematology Am Soc Hematol Educ Program. 2006;509:123
- Krutzik et al. Nat Methods. 2006;3:361
- Lee et al. J Virol. 2008;82:3702
- Yao et al. Proc Natl Acad Sci USA. 2006;103:1000
- Fleisher et al. Clin Immunol. 1999;90:425
- Aboudola et al. Am J Surg Pathol. 2007;31:233
- Martini et al. Am J Clin Pathol. 2008;129:472
- Zuluaga Tora et al. Br J Haematol. 2007;139:31
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