Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling

Background

Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/Principal Findings

We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/Significance

This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.     

Molecular profiling of tyrosine kinases in normal and cancer cells

As the post-genome era is approaching, with vast amount of sequence information available and new technology developed, scientists are presented with opportunities to explore in simple analysis the structure and expression pattern of not just a single gene, but of an entire family of genes, if not the entire genome. The concept of molecular profiling or expression array has thus emerged. The need to simultaneously see all genes in the same family is obvious under the precept of the combinatorial process being an underlying principle of complex biological systems: no gene exists in isolation, for virtually every molecule participates in intermolecular interactions. The activation of receptor tyrosine kinases through homo or hetero-dimerization is the prototypic example. In this review, a tyrosine kinase profile technique and its application to studying the expression of tyrosine kinases and the identification of novel kinases will be discussed. This serves as an introduction to the several interesting papers published in this special kinase issue of theJournal of Biomedical Sciences, using this technique. A new simplified approach, kinase display, which is an extension of the profiling method and requires only restriction digestion and gel analysis will also be introduced.     

Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation

Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.     

Oncostatin M suppresses EGF-mediated protein tyrosine phosphorylation in breast cancer cells

The effect of oncostatin M (OM) on epidermal growth factor (EGF)-mediated protein tyrosine phosphorylation in an infiltrating ductal breast carcinoma cell line, H3922, was investigated by Western blot analysis. Pretreatment of H3922 cells with OM for 72 h suppressed EGF-stimulated protein tyrosine phosphorylation signals by 77%. Interestingly, pretreatment with OM for 6 or 48 h had little effect on these signals. EGF-mediated tyrosine phosphorylation of EGF receptor (EGFR) was suppressed by 55% in 72-h OM pretreated H3922 cells. No reduction in EGFR protein expression was detected in these cells. Flow cytometric analysis verified that OM does not suppress EGFR expression. The effect of OM could not be attributed to induction of protein tyrosine phosphatases. An H3922 subclone cell line, designated H3922-8, was found to exhibit no proliferative response to treatment with EGF. However, EGF-mediated protein tyrosine phosphorylation was detected in these cells. Radioligand EGF binding studies comparing H3922 to H3922-8 cells indicated that the clonal cells apparently lack high affinity EGF receptors. The mechanism by which OM suppresses EGF-mediated tyrosine phosphorylation has not been completely characterized. However, the suppressive effect occurs regardless of whether the cells are acutely responsive (H3922) or virtually unresponsive (H3922-8) to EGF stimulation of cell growth.     

Reduced tyrosine phosphorylation in polyamine-starved cells.

Onset of cell proliferation is associated with enhanced turnover of the polyamines putrescine, spermidine, and spermine, particularly evident in the massive increase in the activity of the rate-limiting enzyme in their production, ornithine decarboxylase (ODC). The physiological functions of these polyamines, however, have remained unclear. Here we report that treatment of LSTRA cells for 2-18 h with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreased the amount of phosphotyrosine in several cellular substrates including the T cell protein tyrosine kinase p56lck. No reductions in the amount of p56lck, overall synthesis of protein and DNA, or cell viability were observed until much later. DFMO did not affect the catalytic activity of p56lck in vitro and the activity of p56lck immunoprecipitated from DFMO-treated cells was unaltered. Addition of putrescine, the reaction product of ODC, completely reversed the effect of DFMO on tyrosine phosphorylation. Finally, we provide evidence that polyamines reduce the activity of cellular protein tyrosine phosphatases toward endogenous substrates. Our results suggest that polyamines may influence the extent of tyrosine phosphorylation during cell proliferation and malignant transformation, perhaps by modulating the rate of dephosphorylation of specific target proteins.     

A method for profiling the phosphorylation state of tyrosine protein kinases

Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.     

Queuine mediated inhibition in phosphorylation of tyrosine phosphoproteins in cancer

Protein phosphorylation or dephosphorylation is the most important regulatory switch of signal transduction contributing to control of cell proliferation. The reversibility of phosphorylation and dephosphorylation is due to the activities of kinases and phosphatase, which determine protein phosphorylation level of cell under different physiological and pathological conditions. Receptor tyrosine kinase (RTK) mediated cellular signaling is precisely coordinated and tightly controlled in normal cells which ensures regulated mitosis. Deregulation of RTK signaling resulting in aberrant activation in RTKs leads to malignant transformation. Queuine is one of the modified base of tRNA which participates in down regulation of tyrosine kinase activity. The guanine analogue queuine is a nutrient factor to eukaryotes and occurs as free base or modified nucleoside queuosine into the first anticodon position of specific tRNAs. The tRNAs are often queuine deficient in cancer and fast proliferating tissues. The present study is aimed to investigate queuine mediated inhibition in phosphorylation of tyrosine phosphorylated proteins in lymphoma bearing mouse. The result shows high level of cytosolic and membrane associated tyrosine phosphoprotein in DLAT cancerous mouse liver compared to normal. Queuine treatments down regulate the level of tyrosine phosphoproteins, which suggests that queuine is involved in regulation of mitotic signaling pathways.     

Heat shock induces protein tyrosine phosphorylation in cultured cells

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.     

Endothelin rapidly stimulates tyrosine phosphorylation in osteoblast-like cells.

The mitogenic activity of endothelin (ET) was studied in osteoblast-like cells, MC3T3-E1. [3H] Thymidine incorporation induced by ET was markedly lower than that of platelet-derived growth factor (PDGF). ET synergistically stimulated [3H] thymidine incorporation induced by PDGF with an apparent ED50 value of 2.5 nM. Treatment of MC3T3-E1 cells with ET and subsequent immunoblotting of the cell extracts with antiphosphotyrosine antibodies followed by labeling with [125I] protein A resulted in the identification of several phosphotyrosine-containing proteins. The intensity of these labeled phosphoproteins significantly increased when the cells were treated with a combination of ET and PDGF. Genistein, an inhibitor of tyrosine kinases, blocked [3H] thymidine incorporation as well as protein tyrosine phosphorylation stimulated by either ET, PDGF or the combination of ET and PDGF. These findings suggest that tyrosine phosphorylation could play a role in the comitogenic activity of ET in osteoblast-like cells.     

Effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells

Tyrosine nitration by peroxynitrite can affect signal transduction pathways involving tyrosine phosphorylation. The present study was undertaken to investigate the effects of peroxynitrite-induced protein tyrosine nitration on insulin-stimulated tyrosine phosphorylation in HepG2 cells. We show here that exposure of HepG2 cells to peroxynitrite led to a dose-dependent increase in tyrosine nitration of cellular proteins, mainly membrane and nuclear proteins. Furthermore, peroxynitrite induced differential responses in tyrosine phosphorylation of membrane proteins as well as cytosolic proteins according to peroxynitrite concentrations used. Our findings indicate at low concentrations peroxynitrite upregulates the insulin signaling and may operate as a signaling molecule, but at higher concentrations peroxynitrite downregulates the insulin signaling and may be involved in insulin resistance, suggesting peroxynitrite plays a dual role in regulation of the insulin signaling.     

Epigenomic profiling of cancer cells

DNA methylation, post-translational modifications of histones and high order organization of chromatin in cell nuclei are the components of the epigenome. Epigenetic regulation of gene expression is specific for each cell type, within different tissues, according to stages of development and (in the adult organism) of differentiation. Almost invariably, this regulation is altered in disease states, including cancer. The complete understanding of the identity of the epigenome of cancer has been so far hampered, due to the technical limitations and costs of the genome-wide analyses required. The recent development of next generation sequencing (NGS) technologies, however, holds the promise of fast, reliable and cost-effective analyses. Here we review the main approaches employed thus far to identify altered epigenetic patterns in cancer cells, and analyse how they are predicted to evolve in the scenario of the ultra high-throughput (UHT) screenings.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Protein kinase CK2 catalyzes tyrosine phosphorylation in mammalian cells

Protein kinase CK2 exhibits oncogenic activity in mice and is over-expressed in a number of tumors or leukemic cells. On the basis of its amino acid sequence and a wealth of experimental information, CK2 has traditionally been classified as a protein serine/threonine kinase. In contrast to this traditional view of CK2, recent evidence has shown that CK2 can also phosphorylate tyrosine residues under some circumstances in vitro and in yeast. In this study, we provide definitive evidence demonstrating that CK2 also exhibits tyrosine kinase activity in mammalian cells. Tyrosine phosphorylation of CK2 in cells and in CK2 immunoprecipitates is dependent on CK2 activity and is inhibited by the CK2 selective inhibitor 4,5,6,7-tetrabromobenzotriazole. Examination of phosphotyrosine profiles in cells reveals a number of proteins, including CK2 itself, which exhibit increased tyrosine phosphorylation when CK2 levels are increased. Peptide arrays to evaluate the specificity determinants for tyrosine phosphorylation by CK2 reveal that its specificity for tyrosine phosphorylation is distinct from its specificity for serine/threonine phosphorylation. Of particular note is the requirement for an aspartic acid immediately C-terminal to the phosphorylatable tyrosine residue. Collectively, these data provide conclusive evidence that CK2 catalyzes the phosphorylation of tyrosine residues in mammalian cells, a finding that adds a new level of complexity to the challenge of elucidating its cellular functions. Furthermore, these results raise the possibility that increased CK2 levels that frequently accompany transformation may contribute to the increased tyrosine phosphorylation that occurs in transformed cells.     

PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells

A phosphatidylinositol (PI) kinase activity associated with certain protein tyrosine kinases important in cell proliferation phosphorylates the 3' hydroxyl position of PI to produce phosphatidylinositol-3-phosphate (PI-3-P). Here we report that, in addition to PI-3' kinase activity, anti-phosphotyrosine (alpha-P-tyr) immunoprecipitates from platelet-derived growth factor (PDGF)-stimulated smooth muscle cells (SMC) contain lipid kinase activities that utilize the substrates phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). These activities are absent in alpha-P-tyr immunoprecipitates from quiescent SMC. The product of PI-4-P phosphorylation appears to be phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), a lipid not previously reported. The product of PI-4,5-P2 phosphorylation is phosphatidylinositol-trisphosphate (PIP3). PI-3-P was detected in quiescent SMC and increased only slightly in response to PDGF. PIP3 and the putative PI-3,4-P2 appeared only after the addition of mitogen. Both the temporal production of these novel phospholipids after PDGF stimulation and the observation of the enzymatic activities that produce them in alpha-P-tyr immunoprecipitates suggest that these phospholipids are excellent candidates for mediators of the PDGF mitogenic response.     

Posttranslational regulation of cyclooxygenase by tyrosine phosphorylation in cerebral endothelial cells

Endothelium-derived cyclooxygenase (COX) products regulatecerebral vascular tone in newborn pigs. Both COX-1 and COX-2 are constitutively expressed in endothelial cells from newborn pig cerebralmicrovessels. We investigated the role of protein phosphorylation inthe regulation of COX activity. The protein tyrosine phosphatase (PTP)inhibitors phenylarsine oxide, vanadate, and benzylphosphonic acidrapidly stimulated COX activity, whereas the protein tyrosine kinaseinhibitors, genistein and tyrphostins, inhibited it. Protein synthesisinhibitors did not reverse the stimulation of COX activity evoked byPTP inhibitors. Similar changes were observed in other vascular cellsfrom newborn pigs that also express COX-1 and COX-2 (cerebralmicrovascular smooth muscle cells and aortic endothelial cells) but notin human umbilical vein endothelial cells or Swiss 3T3 fibroblasts thatexpress COX-1 only. Tyrosine-phosphorylated proteins wereimmunodetected in endothelial cell lysates. COX-2 immunoprecipitatedfrom ³²P-loaded endothelial cellsincorporated ³²P that wasincreased by PTP inhibitors. COX-2, but not COX-1, was detected inendothelial fractions immunoprecipitated with anti-phosphotyrosine.These data indicate that tyrosine phosphorylation posttranslationallyregulates COX activity in newborn pig vascular cells and that COX-2 isa substrate for phosphorylation.

     

Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells

The role of fibroblast growth factor-2 (FGF-2) in maintaining undifferentiated human embryonic stem cells (hESC) was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs) was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.     

Growth inhibition and protein tyrosine phosphorylation in MCF 7 breast cancer cells by a novel K vitamin

We have now found that the most potent, Cpd 5 [2-(2-mercaptoethanol)-3-methyl-1, 4-napthoquinone], inhibits growth of doxorubicin-resistant and doxorubicin-sensitive breast cancer cells (MCF 7r and MCF 7w) in culture. Growth inhibition by Cpd 5 was antagonized by the thiol antioxidants glutathione and cysteine, but not by catalase or superoxide dismutase, suggesting that growth inhibition is probably via conjugation of cellular thiols. In support of this, we found that Cpd 5 inhibited the activity of thiol containing cellular protein tyrosine phosphatase (PTP) enzyme, with consequent induction of various tyrosine phosphoproteins, but not serine or tyrosine phosphoproteins. The tyrosine phosphorylation was also inhibited by exogenous glutathione or cysteine and could be enhanced by depletion of cellular glutathione by BSO. This effect of Cpd 5 on protein tyrosine phosphorylation was highly selective, however. Tyrosine phosphorylation of EGF-R, Erb-B2, and ERK1/2 was increased, but not that of Insulin-R or JNK. ERK1/2 tyrosine phosphorylation and growth inhibition increased with increasing concentrations of Cpd 5. Furthermore, suppression of Cpd 5-mediated ERK1/2 phosphorylation by an ERK-kinase inhibitor antagonized growth inhibition. These results suggest a strong correlation between ERK1/2 phosphorylation by Cpd 5 and growth inhibition. This novel K-vitamin analog thus inhibits MCF 7 cell growth and induces selective protein tyrosine phosphorylation.