Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications

Krutzik, Peter O.; Irish, Jonathan M.; Nolan, Garry P.; Perez, Omar D.

doi:10.1016/j.clim.2003.11.009

Cited by 297 publications

(225 citation statements)

References 70 publications

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“…Indeed, this approach allows the use of less cells when compared with Western blot and is rapidly becoming the preferred technique for the analysis of drug resistance-linked proteins 27 and phosphorylated proteins [28][29][30] in AML. Therefore, a very critical issue concerned the specificity of the antibodies used in our study.…”

Section: Specificity Of the Antibodiesmentioning

confidence: 99%

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

et al. 2007

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A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.

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Section: Specificity Of the Antibodiesmentioning

confidence: 99%

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

et al. 2007

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“…Given the abnormalities in T cell proliferation, we examined endogenous signaling pathways associated with activation, specifically phosphorylation of linker of activated T cells (LAT). We analyzed the pattern of phospho-LAT (pLAT) in OTII T cells stimulated with DCs at day 3 and day 6 of coculture by using phosphospecific flow cytometry (PhosphoFlow) techniques (17)(18)(19). Between the coculture groups, we specifically compared T cells of equivalent cell size, to enhance our resolution of activation-induced phosphorylation (Fig.…”

Section: Inhibition Of Sema6d Reduces a Late Phase Of T Cell Activatimentioning

confidence: 99%

Semaphorin 6D regulates the late phase of CD4 ⁺ T cell primary immune responses

O’Connor

Eun

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell-cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor-ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.immune regulation ͉ lymphocyte selection ͉ response maturation

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“…Intracellular flow cytometry using phospho-site specific antibodies (Phospho-Flow) has recently evolved as a sophisticated, powerful technology to study phosphorylation signatures in cell lines or subsets of primary cells (5,6). In contrast to Western blot (WB), which allows the detection of phosphorylated proteins in denatured cell lysates, Phospho-Flow phosphorylated analytes are assessed in fixed and permeabilized cells.…”

mentioning

confidence: 99%

Intracellular Phospho‐Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot

Haas¹,

Weckbecker²,

Welzenbach³

2008

Cytometry Pt A

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Phospho-site specific antibodies become increasingly available, enabling the study of signaling events by Western blotting (WB) or intracellular flow cytometry (PhosphoFlow). Here we compared data generated by WB or Phospho-Flow regarding the kinetics and degree of phosphorylation of membrane proximal TCR signaling molecules. Phosphorylation events in Jurkat T cells were triggered by anti-CD3 stimulation (OKT3) or by oxidative stress (H 2 O 2 ) and were analyzed by Phospho-Flow or WB. Both techniques showed that OKT3-or H 2 O 2 -induced, transient phosphorylation of ZAP70 or LAT was dependent on functional Lck. Phospho-Flow data revealed differences in the kinetics and the degree of H 2 O 2 -or OKT3-mediated protein phosphorylation compared with WB data. In addition, using Phospho-Flow we discovered that H 2 O 2 -induced phosphorylation of TCR signaling proteins was inhibited by small molecular weight kinase inhibitors far more potently than OKT3-triggered protein phosphorylation, despite a superior induction of phosphorylation by H 2 O 2 .This finding was confirmed by WB. Interestingly, we identified by Phospho-Flow that, in P116 Jurkat cells lacking ZAP70 protein expression, H 2 O 2 potently triggered the phosphorylation of ZAP70 residues Y493 and Y292 but not Y319. The phosphorylation of these ZAP70 tyrosine residues cells was blocked by an Lck inhibitor, suggesting the existence of an Lck-coupled truncated ZAP70 protein or a novel isoform of ZAP70 in P116 cells. Phospho-Flow is a largely quantitative technology with excellent throughput, highly suited in studying the function or inhibition of TCR signaling pathways and allowing the detection of novel pathway insights. It can serve as a good complement to Western blot analysis. '

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Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications

Cited by 297 publications

References 70 publications

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

Semaphorin 6D regulates the late phase of CD4 ⁺ T cell primary immune responses

Intracellular Phospho‐Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot

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Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications

Cited by 297 publications

References 70 publications

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

Semaphorin 6D regulates the late phase of CD4 + T cell primary immune responses

Intracellular Phospho‐Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot

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Semaphorin 6D regulates the late phase of CD4 ⁺ T cell primary immune responses