Tyrosine Kinase Activity In Cells Research Articles

Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling.

In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases is critical for revealing and therapeutically manipulating mechanisms of immune regulation. Quantitative analysis of the amplitude and kinetics of these effects is becoming ever more accessible experimentally and increasingly important for predicting complex downstream effects of therapeutics and for building computational models. Secondarily, quantitative analysis is increasingly expected by reviewers and journal editors, and statistical analysis of biological replicates can bolster claims of experimental rigor and reproducibility. Here we outline methods for perturbing tyrosine kinase activity in cells and quantifying protein phosphorylation in lysates and immunoprecipitates. The immunoblotting techniques are a guide to probing the dynamics of protein abundance, protein–protein interactions, and changes in post‐translational modification. Immunoprecipitated protein complexes can also be subjected to targeted mass spectrometry to probe novel sites of modification and multiply modified or understudied proteins that cannot be resolved by immunoblotting. Together, these protocols form a framework for identifying the unique contributions of tyrosine kinases to cell activation and elucidating the mechanisms governing immune cell regulation in health and disease. © 2020 The Authors. Basic Protocol 1: Quantifying protein phosphorylation via immunoblotting and near‐infrared imaging Alternate Protocol: Visualizing immunoblots using chemiluminescence Basic Protocol 2: Enriching target proteins and isolation of protein complexes by immunoprecipitation Support Protocol: Covalent conjugation of antibodies to functionalized beads Basic Protocol 3: Quantifying proteins and post‐translational modifications by targeted mass spectrometry

Assays for tyrosine phosphorylation in human cells.

Tyrosine kinases are important for many cellular processes and disruption of their regulation is a factor in diseases like cancer, therefore they are a major target of anticancer drugs. There are many ways to measure tyrosine kinase activity in cells by monitoring endogenous substrate phosphorylation, or by using peptide substrates and incubating them with cell lysates containing active kinases. However, most of these strategies rely on antibodies and/or are limited in how accurately they model the intracellular environment. In cases in which activity needs to be measured in cells, but endogenous substrates are not known and/or suitable phosphospecific antibodies are not available, cell-deliverable peptide substrates can be an alternative and can provide information on activation and inhibition of kinases in intact, live cells. In this chapter, we review this methodology and provide a protocol for measuring Abl kinase activity in human cells using enzyme-linked immunosorbent assay (ELISA) with a generic antiphosphotyrosine antibody for detection.

Phase 1 dose-escalation, pharmacokinetic, and cerebrospinal fluid distribution study of TAK-285, an investigational inhibitor of EGFR and HER2

Summary Introduction This phase 1 study assessed safety, maximum tolerated dose (MTD), pharmacokinetics, cerebrospinal fluid (CSF) distribution, and preliminary clinical activity of the receptor tyrosine kinase inhibitor TAK-285. Methods Patients with advanced, histologically confirmed solid tumors and Eastern Cooperative Oncology Group performance status ≤2 received daily oral TAK-285; daily dose was escalated within defined cohorts until MTD and recommended phase 2 dose (RP2D) were determined. Eleven patients were enrolled into an RP2D cohort. Blood samples were collected from all cohorts; CSF was collected at pharmacokinetic steady-state from RP2D patients. Tumor responses were assessed every 8 weeks per Response Evaluation Criteria in Solid Tumors. Results Fifty-four patients were enrolled (median age 60; range, 35–76 years). The most common diagnoses were cancers of the colon (28 %), breast (17 %), and pancreas (9 %). Escalation cohorts evaluated doses from 50 mg daily to 500 mg twice daily; the MTD/RP2D was 400 mg twice daily. Dose-limiting toxicities included diarrhea, hypokalemia, and fatigue. Drug absorption was fast (median time of maximum concentration was 2–3 h), and mean half-life was 9 h. Steady-state average unbound CSF concentration (geometric mean 1.54 [range, 0.51–4.27] ng/mL; n = 5) at the RP2D was below the 50 % inhibitory concentration (9.3 ng/mL) for inhibition of tyrosine kinase activity in cells expressing recombinant HER2. Best response was stable disease (12 weeks of nonprogression) in 13 patients. Conclusions TAK-285 was generally well tolerated at the RP2D. Distribution in human CSF was confirmed, but the free concentration of the drug was below that associated with biologically relevant target inhibition.

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP–Cy3 donor–acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1–4] and concomitant activation of its intracellular tyrosine kinase domain [5–7]. Trans-autophosphorylation of the receptor [8,9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.

Functional Characterization of an Epidermal Growth Factor Receptor/RET Chimera

The RET (recombined in transfection) gene encodes a receptor tyrosine kinase homolog involved in innervation of the gut and renal development. A chimeric epidermal growth factor receptor (EGFR)/RET receptor was constructed which contained the extracellular and transmembrane domains of the EGF receptor fused to the intracellular domain of RET. This construct was expressed in NIH 3T3 cells, and the functional properties of the receptor were characterized and compared with those of the wild type EGF receptor. Whereas the EGF receptor exhibited both high and low affinity binding sites for 125I-EGF, the EGFR/RET chimera exhibited only low affinity binding of 125I-EGF. The chimera was able to internalize EGF more rapidly than the wild type EGF receptor and recycled to the cell surface at twice the rate of the EGF receptor. Pulse-chase experiments indicated that EGF stimulated the degradation of the wild type EGF receptor but had no effect on the rate of degradation of the EGFR/RET receptor. The combination of increased recycling and decreased degradation resulted in the relatively inefficient down-regulation of the EGFR/RET chimera. Incubation of cells expressing the wild type EGF receptor with phorbol 12-myristate 13-acetate led to a reduction in 125I-EGF binding and a loss in EGF-stimulated tyrosine phosphorylation. However, phorbol 12-myristate 13-acetate treatment had only a limited effect on EGF binding and EGF-stimulated tyrosine kinase activity in cells expressing EGFR/RET chimeras. These findings suggest that the ret tyrosine kinase is not regulated by many of the common mechanisms used to terminate signaling via growth factor receptors. Such persistent activation of the Ret tyrosine kinase may be relevant to the physiological function of Ret in cells that normally express this growth factor receptor.

Arachidonic Acid Induces c-jun Gene Expression in Stromal Cells Stimulated by Interleukin-1 and Tumor Necrosis Factor-α: Evidence for a Tyrosine-Kinase-Dependent Process

Abstract We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O- tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c- jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)- induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c- jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF- alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic acid induces c-jun gene expression in stromal cells stimulated by interleukin-1 and tumor necrosis factor-alpha: evidence for a tyrosine-kinase-dependent process

We have previously shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the murine stromal cell line +/+.1-LDA 11 involves activation of phospholipase A2 (PLA2). Furthermore, induction of GM-CSF gene expression due to release of arachidonic acid as a result of PLA2 activation was mediated by the transcriptional factor c-jun. In the present study, we have investigated the potential mechanism involved in the induction of c-jun gene expression by arachidonic acid. Arachidonic acid induced transcription of c-jun mRNA. Downregulation of protein kinase C (PKC) by chronic exposure of stromal cells to the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 400 nmol/L) did not effect c-jun expression induced by arachidonate. Moreover, pretreatment of cells with the PKC inhibitor, calphostin C (1 mumol/L), caused a marked decrease of c-jun expression induced by TPA, but had no influence on c-jun expression induced by arachidonate. To explore the hypothesis that a tyrosine kinase signalling pathway, independent of PKC activation, was involved in arachidonate-induced c-jun expression, stromal cells were pretreated with the protein tyrosine kinase inhibitor, genistein, before challenge with arachidonic acid. Arachidonate 50 mumol/L)-induced c-jun expression was inhibited, in a dose- and time-dependent manner, by genistein. Genistein similarly inhibited c-jun expression in stromal cells exposed to IL-1 (500 U/mL) plus TNF-alpha (500 U/mL). The potential role of a tyrosine kinase pathway in arachidonate-mediated c-jun expression was further investigated by assaying the tyrosine kinase activity of cells challenged with arachidonic acid, IL-1, and TNF-alpha. Exposure of stromal cells to arachidonic acid induced a 2.1-fold increase in intracellular tyrosine kinase activity determined by phosphorylation of the synthetic peptide, raytide, in the presence of [gamma-32P]-ATP. Similarly, IL-1 and TNF-alpha induced 1.7- and 2.4-fold increases in tyrosine protein kinase activity, respectively. The effect of arachidonic acid on tyrosine kinase activity was inhibited by genistein and was enhanced by sodium vanadate. The increase of protein tyrosine kinase activity detected in arachidonate-stimulated cells was associated, in a dose- and time-dependent fashion, with tyrosine phosphorylation of 240-, 40-, and 29-kD substrates. These results are consistent with the hypothesis that a tyrosine phosphorylation process is triggered by arachidonate as an early event in the signalling pathway that leads to increased expression of c-jun.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory Effect of Interleukin-4 on the In Vitro Growth of Ph1-Positive Acute Lymphoblastic Leukemia Cells

Abstract We investigated the effect of recombinant human interleukin-4 (rhIL-4) on the in vitro growth of human leukemia cells in liquid culture and 3H- thymidine incorporation and found inhibitory effects on the growth of leukemic cells from patients with Ph1-positive acute lymphoblastic leukemia (Ph1 ALL) and three Ph1 ALL cell lines. However, no inhibitory effects were seen in Ph1-positive leukemic cell lines derived from patients with chronic myelogenous leukemia in blast crisis and various types of Ph1-negative leukemia cells, including B-lineage leukemia cells. In a flow cytometry assay of IL-4 receptor (IL-4R), all three Ph1-positive ALL cell lines showed the presence of IL-4R on their cell surfaces, and the IL-4-dependent inhibition on the growth of Ph1- positive ALL cells was abrogated by the addition of either monoclonal or polyclonal antibodies against rhIL-4. Other cytokines, including IL- 2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and IL-6, showed no inhibitory effects on the growth of Ph1-ALL cells, but tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN)-alpha, -beta, and -gamma displayed slight inhibitory effects in a high concentration. The growth inhibition induced by rhIL- 4 in the Ph1-positive ALL cells was not abrogated by the addition of antibodies against either IFN-gamma or TNF-alpha. Furthermore, these cells showed no significant production of IFN-alpha, -beta, or -gamma or TNF-alpha after exposure to rhIL-4, thus indicating that the growth inhibition of Ph1-positive ALL cells by rhIL-4 is not associated with IL-4-stimulating production of these factors. rhIL-4 caused significant inhibition of the tyrosine kinase activity in these Ph1-positive ALL cells, similar to Herbimycin A, an inhibitor of tyrosine kinase that inhibited the tyrosine kinase activity in these cells. Our finding suggests that the clinical evaluation of rhIL-4 may offer promising therapeutic possibilities for patients with Ph1-positive ALL.

Inhibitory effect of interleukin-4 on the in vitro growth of Ph1- positive acute lymphoblastic leukemia cells

We investigated the effect of recombinant human interleukin-4 (rhIL-4) on the in vitro growth of human leukemia cells in liquid culture and 3H-thymidine incorporation and found inhibitory effects on the growth of leukemic cells from patients with Ph1-positive acute lymphoblastic leukemia (Ph1 ALL) and three Ph1 ALL cell lines. However, no inhibitory effects were seen in Ph1-positive leukemic cell lines derived from patients with chronic myelogenous leukemia in blast crisis and various types of Ph1-negative leukemia cells, including B-lineage leukemia cells. In a flow cytometry assay of IL-4 receptor (IL-4R), all three Ph1-positive ALL cell lines showed the presence of IL-4R on their cell surfaces, and the IL-4-dependent inhibition on the growth of Ph1-positive ALL cells was abrogated by the addition of either monoclonal or polyclonal antibodies against rhIL-4. Other cytokines, including IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, and IL-6, showed no inhibitory effects on the growth of Ph1-ALL cells, but tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN)-alpha, -beta, and -gamma displayed slight inhibitory effects in a high concentration. The growth inhibition induced by rhIL-4 in the Ph1-positive ALL cells was not abrogated by the addition of antibodies against either IFN-gamma or TNF-alpha. Furthermore, these cells showed no significant production of IFN-alpha, -beta, or -gamma or TNF-alpha after exposure to rhIL-4, thus indicating that the growth inhibition of Ph1-positive ALL cells by rhIL-4 is not associated with IL-4-stimulating production of these factors. rhIL-4 caused significant inhibition of the tyrosine kinase activity in these Ph1-positive ALL cells, similar to Herbimycin A, an inhibitor of tyrosine kinase that inhibited the tyrosine kinase activity in these cells. Our finding suggests that the clinical evaluation of rhIL-4 may offer promising therapeutic possibilities for patients with Ph1-positive ALL.

Effect of granulocyte-macrophage colony-stimulating factor and interleukin 3 on the v-src oncogene. Inhibition of tyrosine kinase activity in the absence of changes in gene expression.

Three cloned murine interleukin 3 (IL-3)-dependent cell lines have been converted to interleukin 2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF) growth-dependent states. FD.C/1 32Dcl-23 and GM cells grown and maintained as IL-3-dependent cell lines, and cells grown with GM-CSF have been infected with a murine recombinant retrovirus containing the v-src oncogene, and grown as lymphokine-independent cell lines. There is a significant increase in tyrosine kinase activity in cells which become lymphokine-independent. FD.C/1 and 32Dcl-23 cells maintained as IL-2-dependent cells lines and infected with the same virus did not grow as IL-2-independent cells. The lymphokine-independent cells FD.C/1src, 32Dsrc, and GMsrc all expressed high levels of tyrosine kinase activity, ranging from 5- to 20-fold more than levels measured in virus-infected cell lines maintained as IL-2-dependent cells. The exposure of FD.C/1src and 32Dsrc cells to IL-3, and GMsrc cells to IL-3 or GM-CSF, resulted in significant decreases in tyrosine kinase activity. These changes were rapidly reversed by removal of IL-3 or GM-CSF from these cells. However, the synthesis of v-src-specific RNA was not affected by the presence of IL-3 or GM-CSF in these cell lines. The biochemical pathways activated by IL-3 or GM-CSF inhibit the activity of the tyrosine kinase encoded by the v-src oncogene without altering gene transcription.