Kinase Activity Profiling Research Articles

Abstract 797: Kinase activity profiling of tumor tissues of different origin

Abstract Introduction: Many new anti-cancer drugs target kinase activity. Unfortunately, methods to monitor the drug effects at the enzymatic level in patient derived tumor tissue are lacking. Here, a novel molecular profiling method for application in biomarker discovery is presented. This method is based on measuring kinase activities in tumor tissue extracts, and involves assessment of inhibition by a drug of interest. This ex vivo activity-based approach is enabled by dynamic peptide microarrays. These arrays comprise of peptides, which are known substrates for phosphorylation by protein kinases. Here we investigated the applicability of this approach in the classification of multiple tumors of different origin. Method: Patient-derived fresh frozen tissues of different tumor types (24 breast cancer (BC), 14 non-small cell lung cancer (NSCLC), 12 renal cell carcinomas (RCC)) were used for extraction of total protein in lysis buffer. Dependent on the tissue source, 6 slices of 10 µm were sufficient for 20-100 microarray analyses. Equal amounts of total protein were analyzed for kinase activity on dynamic PamChip peptide microarrays. These arrays comprised 144 or 256 different peptides. A PamChip96 plate format was used, enabling 96 microarray incubations per run. Protein amounts ranging from 1 − 5 µg were used per microarray analysis. Tumor extracts were analysed in the presence and absence of kinase inhibitor drugs (RCC: sorafenib and sunitinib, NSCLC: gefitinib). Results: We show here that from different tumors kinase activity profiles could be generated reproducibly (10-15% CV for selected peptides). Control experiments showed protein concentration and ATP dependency. In the RCC experiments different phosphorylation profiles were obtained when the non-tumor tissue was compared to tumor tissue derived from the same patient, showing higher kinase activities in the latter. In addition, inhibitor dependent modulation of the peptide phosphorylations was observed (sorafenib vs. sunitinib). Furthermore, differential kinase activity profiles were obtained that could be correlated to different patient subgroups (BC) or clinical data (long vs. short term survivors; NSCLC). Conclusion: A novel molecular profiling approach was successfully applied for classification of different tumors. This approach is based on detection of kinase activities as well as inhibition of kinase activity in tumor tissues. Application of this method in the discovery of biomarkers for diagnosis, prognosis or prediction of drug response is foreseen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 797.

Abstract 5577: Global analysis of protein kinase activity during hematopoietic differentiation

Abstract Cell proliferation and differentiation are highly regulated processes, and the deregulation of these processes can lead to a number of diseases including cancer. Protein kinases are key regulators of cell proliferation and differentiation, and consequently, potential targets for a number of oncology indications. A detailed understanding of the changes in kinase activity/expression during cell proliferation and differentiation may be one of the keys to the identification of novel biomarkers and targets for the diagnosis and treatment of these diseases. The HL-60 cell line is promyeloblastic, and protocols for the differentiation of these cells into either macrophages by 12-O-tetradecanoylphorbol-13-acetate (PMA), or granulocytes by retinoic acid (RA) are well-established. Importantly, HL-60 cell differentiation is a model system for the treatment of myelocytic leukemia, and identifying the mechanisms that induce terminal differentiation of these cells may reveal effective targets for treatment of this disease. With this in mind, we evaluated the kinase activity and expression profiles of HL-60 cells throughout the differentiation process using desthiobiotin acyl phosphate ATP and ADP probes and liquid chromatography-mass spectrometry. Greater than 150 protein kinases in resting, RA, and PMA HL-60 cells were analyzed. Significant changes in activity/expression were seen for a number of protein kinases in both differentiation pathways. These changes in kinase activity were generally consistent with changes in protein expression levels, as determined by Western blot. In both RA and PMA-induced HL60 differentiation, the kinetics for the changes in activity of the kinases was determined, and the results suggest that there is a well-defined sequence during which these changes occur. In summary, using activity based protein profiling, we identified a subset of kinases whose activity changed significantly during RA and PMA-induced HL-60 differentiation. Importantly, this methodology is widely applicable to other cellular systems and disease models, and has applications in the identification of biomarkers and targets for the management of these diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5577.

Abstract 2386: High-throughput assessment of the VEGFR2 kinase activity profile in pediatric brain tumor tissue

Abstract For enhanced tumor cell survival and progression, tumors depend on angiogenesis. Vascular endothelial growth factor receptor (VEGFR) signaling plays a major part in this process. Previously we generated a comprehensive description of the biological tyrosine kinase activity present in pediatric brain tumor tissue lysates by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates (Sikkema et al., Cancer Res 2009). The results suggested the presence of activated VEGF receptors in pediatric pilocytic astrocytoma tissue. We hypothesized that this detected VEGFR activity reflects the angiogenic status of the tumor endothelium. In this study we demonstrated that VEGFR2 expression is not present in a pilocytic astrocytoma cell line. Moreover, proximity ligation assays on tumor cryosections showed VEGFR2 phosphorylation, primarily localized on endothelial cells. qRT-PCR analysis of endothelial markers and VEGFRs showed a 10 to 30-fold enrichment of VEGFR2 expression on laser microdissected endothelium compared to whole tumor. Hence, we conclude that the endothelial cells are the high producers of VEGFR2 kinase activity in these tumors and therefore responsible for the VEGFR2 kinase activity that we observed in our kinase activity screens. In conclusion, potential VEGFR kinase substrates present on the peptide microarray are adequately sensitive to pick up enzymatic activity which is present in only a subset of the total cell population within solid tumors such as pilocytic astrocytoma. In the near future, high throughput measurement of enzymatic activity of key players in angiogenesis could make a rapid assessment of the angiogenic profile of a tumor possible. This could serve as an indicator of anti-angiogenic treatment response and result in more personalized anti-angiogenic cancer treatment strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2386.

Abstract 650: Response prediction to a multitargeted tyrosine kinase inhibitor by profiling serine/threonine kinase activity and inhibition

Abstract Introduction: Since treatment in oncology is rarely effective in all patients, stratification of patients with innate resistance or monitoring the emergence of acquired resistance will prevent patients being treated with ineffective drugs and will further the development of personal medicine. Several examples of such prediction methods have been reported, based on gene expression or presence of marker proteins. For kinase inhibitors, that target active kinases, it makes sense to monitor their effect on kinase activity ex vivo on a patients own tumor tissue. For the prediction of cell line response to a multityrosine kinase inhibitor (MTKI) in a proliferation assay, the feasibility of this approach has been shown, using tyrosine kinase activity profiling on a dynamic peptide microarray. Here we investigated the possibility of response prediction by profiling serine/threonine kinase activities. Methods 24 Cancer cell lines were cultured in the presence of a multi-tyrosine kinase inhibitor and the IC50 determined in a proliferation assay. Lysates from untreated cells were incubated in triplicate on a PamChip microarray comprising 140 ser/thr containing peptides in the presence and absence of hydroxystaurosporin or flavopiridol. Peptide phosphorylation by the lysates was monitored using a mixture of phosphoserine and phosphothreonine detecting antibodies. Ratios of inhibited/non-inhibited signal intensities were calculated for each peptide and correlated to the proliferation assay data. A peptide subset that gave ATP dependent signals (p<0.001) was used for classification by partial least square discriminant analysis (PLS-DA). Results. As shown previously, inhibition of tyrosine phosphorylation by MTKI was able to predict response or resistance of cells both in a proliferation assay and in xenografts1). Here, phosphorylation by ser/thr kinases was investigated. Basal phosphorylation profiles obtained on the PamChip peptide microarray did not correlate with sensitivity in the proliferation assay. Using ratios of phosphorylation in the presence and absence of inhibitor hydroxystaurosporin (a PKC/PDPK inhibitor), a peptide set able to predict response in the proliferation assay was identified. More inhibition was observed in cell lines expressing an activated Akt signaling pathway. When flavopiridol (a CDK inhibitor) was used, no profile predictive of MTKI response was found. Conclusion. Peptide microarrays can be used to predict response to treatment as exemplified for cell lines in a proliferation assay. Prediction of response can be based on inhibition profiles of both tyrosine kinases activity and ser/thr kinase activity. The multiparameter readout of kinase activity links response prediction to activity of relevant signaling pathways by the appropriate choice of kinase inhibitors. 1) Versele et al. Mol Cancer Therapeutics 2009; 8(7), 1846-1855. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 650.

Abstract 5559: Proteomic identification of activated, essential tyrosine kinases in human cancers

Abstract Tyrosine kinases (TKs) are frequently, aberrantly activated in human cancers. Moreover, they could be easily inhibited by small molecules and thereby represent excellent therapeutic targets. Here, our project aims to systematically identify activated, essential TKs in human cancers. In our previously published study, we have developed a high-throughput, multiplex antibody-based assay capable of identifying activated tyrosine kinases regardless of their mode of activation. Using this approach, we profiled 130 established cancer cell lines and 31 primary patient samples. In particular, we found frequent SRC activation in glioblastomas in the absent of genomic alterations. Moreover, we determined SRC essentiality and demonstrated SRC being a relevant target of a FDA-approved drug, dasatinib, in this deadly disease. Since then, we have expanded the Luminex assay to include additional TKs, adaptors and other important signaling molecules. Specifically, the version 2 of this assay is capable of simultaneously analyzing the tyrosine phosphorylation levels on 71 out of the 90 TKs, 9 adaptor proteins, ERK1/2, PLCG1 in the human genome. In addition, we have established separate assays to measure activities of AKT1, GSK3B, p70S6K and RSK1/2. Besides expanding the panel of analytes, we have made significant improvement on assay setup and data analysis. In particular, we have fully automated assay setup in 96-well format. In addition, we have generated negative and positive control samples to enable more accurate background signal estimation and plate-to-plate comparison, respectively. Using the improved version o f the assay, we have profiled 200 additional cancer cell lines and 100 primary tumor specimens. We found that some TKs are frequently activated across multiple malignancies while other are restricted to a particular tumor type. Interestingly, majority of the TKs are activated at very low frequency. Together, these results stress the importance of personalized treatment (i.e. choosing kinase inhibitors based on kinase activation profile of a tumor). In parallel to our effort on identifying activated TKs, we are systematically assessing TK essentiality using the pharmacological approach. Specifically, we have collected 25 small molecules targeting all known oncogenic TKs and are in the process of measuring sensitivities towards these inhibitors in various cancer cell lines. By comparing TK activation status and sensitivity to inhibitors, we have identified potential targets and corresponding drug candidates for treating subsets of cancer patients. Together, we have gained a comprehensive view on aberrant TK activations and downstream signaling in human cancers. Beyond the mechanistic understanding of tyrosine kinase signaling, our study has identified potential targets for devising effective therapeutic strategies for treating cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5559.

Downregulation of Cdc2/CDK1 Kinase Activity Induces the Synthesis of Noninfectious Human Papillomavirus Type 31b Virions in Organotypic Tissues Exposed to Benzo[ a ]pyrene

Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16(INK4) and p27(KIP1), which normally mediate pRb hypophosphorylation. In contrast, p21(WAF1) and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to BaP-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. BaP targeting of CDK1 occurred independently of HPV status, since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.

56LBA Prediction of response to preoperative chemoradiotherapy in rectal cancer by multiplex kinase activity profiling

A fatigue prediction method considering shrinkage cavity, secondary dendrite arm spacing (SDAS) and mean stress level is presented in the paper. Firstly, the casting process of an aluminum alloy wheel is simulated based on ProCAST software. And the data of SDAS and porosity of different parts are predicted based on the solidification process. Then the data mapping algorithm between tetrahedral mesh elements is developed to realize the unidirectional transformation of microcosmic data from a cast model to a static mechanical model. And the radial loading mechanical analysis model of a wheel containing microcosmic information is further established. According to the specific mechanical and fatigue parameters of each node, the fatigue life prediction model is established by Fesafe software. Based on the self-developed Transfer Couple Data (TCD) software, the integrated coupling method of the three software prediction models is realized, and the method is further used to realize a precise prediction of the radial fatigue life of a wheel considering effects of shrinkage cavity, SDAS and mean stress. Compared with the experimental results, after considering the microcosmic influence, the predicted position of the minimum life is unchanged, and the predicted life value is more accurate after considering the microcosmic influence. The proposed method lays a solid foundation of the optimization design and lightweight design of aluminum alloy wheels.

Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinase-mediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies.

PTK787/ZK 222584 inhibits tumor growth promoting mesenchymal stem cells: Kinase activity profiling as powerful tool in functional studies

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been shown to favour tumor growth, suggesting the relevance of pharmaceutical inhibition of MSCs for the treatment of malignancies. We tested the effect of PTK787/ZK 222584 (PTK) on the outgrowth of MSCs from human bone marrow-derived mononuclear cells (MNCs) and the migration and tube formation capacity of MSCs in vitro. PTK dose- dependently inhibited the outgrowth of BM-MSCs from BM-MNCs (LC50 1.12 μM PTK), while hematopoietic colony formation (HCF) was only slightly hampered (13 ± 19% at 1 μM PTK, and stable at ~50% at higher concentrations of PTK). Addition of 10 μM PTK inhibited proliferation of MSCs by 74 ± 6.6% compared to control (p

Phosphate-Affinity Gel Electrophoresis Using a Phos-Tag Molecule for Phosphoproteome Study

Recently, we developed a novel type of phosphate-affinity gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear manganese(II) complex of a phosphate-binding tag nanomolecule, Phos-tag, which enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterparts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the quantitative analysis of protein kinase and phosphatase reactions on a polyacrylamide gel without any special apparatuses, radioactive isotopes, or chemical labels. This review article summarizes four applications of protein phosphorylation profiling using a type of affinity electrophoresis, Mn2+ – Phos-tag SDS-PAGE, as follows: i) in vitro kinase activity profiling for the analysis of the phosphoprotein isotypes derived from various kinase reactions, ii) in vivo kinase activity profiling for the analysis of extracellular signal-dependent protein phosphorylation, iii) in vitro kinase inhibition profiling for the quantitative analysis of a kinase-specific inhibitor, and iv) a two-dimensional mobility-shifting procedure using Mn2+ – Phos-tag SDS-PAGE for the detailed analysis of phosphoprotein isotypes. In addition, we describe the significant advantages, including a higher resolution power for the separation of protein phosphoisotypes compared with the conventional gel-based electrophoresis methods. Protein phosphorylation profiling can provide the basis for understanding the molecular origins of diseases and potentially developing tools toward therapeutic intervention. Therefore, the phosphate-affinity gel electrophoresis methodologies established by using Phos-tag can greatly facilitate the phosphoproteomics for the determination of protein phosphorylation status in life science laboratories worldwide. Keywords: Phos-tag, affinity electrophoresis, protein kinase, protein phosphatase, phosphorylated protein, phosphoproteomics, phosphorylation, signal transduction

Episodic Src activation in uveal melanoma revealed by kinase activity profiling

Background:The RAS/RAF/MEK/ERK pathway is involved in the balance between melanocyte proliferation and differentiation. The same pathway is constitutively activated in cutaneous and uveal melanoma (UM) and related to tumour growth and survival. Whereas mutant BRAF and NRAS are responsible for the activation of the RAS/RAF/MEK/ERK pathway in most cutaneous melanoma, mutations in these genes are usually absent in UM.Methods:We set out to explore the RAS/RAF/MEK/ERK pathway and used mitogen-activated protein kinase profiling and tyrosine kinase arrays.Results:We identified Src as a kinase that is associated with ERK1/2 activation in UM. However, low Src levels and reduced ERK1/2 activation in metastatic cell lines suggest that proliferation in metastases can become independent of Src and RAS/RAF/MEK/ERK signalling. Inhibition of Src led to the growth reduction of primary UM cultures and cell lines, whereas metastatic cell line growth was only slightly reduced.Conclusion:We identified Src as an important kinase and a potential target for treatment in primary UM. Metastasis cell lines seemed largely resistant to Src inhibition and indicate that in metastases treatment, a different approach may be required.

Osteoblast-induced EGFR/ERBB2 signaling in androgen-sensitive prostate carcinoma cells characterized by multiplex kinase activity profiling

Bone metastases in prostate cancer are predominantly osteoblastic. To study regulatory mechanisms underlying the establishment of prostate cancer within an osteoblastic microenvironment, human androgen-sensitive prostate carcinoma cells (LNCaP) were treated with culture medium conditioned by human osteoblast-derived sarcoma cells (OHS), and activated signalling pathways in the carcinoma cells were analyzed using microarrays with tyrosine kinase substrates. Network interaction analysis of substrates with significantly increased phosphorylation levels revealed that signalling pathways mediated by EGFR and ERBB2 were activated in LNCaP cells under OHS influence but also by androgen treatment. Activation of EGFR/ERBB2 signalling was also found in LNCaP cells in cocultures with OHS cells or osteoblastic cells that had been differentiated from human mesenchymal stem cells. Our experimental data suggests osteoblast-directed induction of signalling activity via EGFR and ERBB2 in prostate carcinoma cells and may provide a rationale for the use of EGFR or ERBB2 inhibition in systemic prevention or treatment of metastatic prostate cancer in the androgen-sensitive stage of the disease.

TiO2-assisted silver enhanced biosensor for kinase activity profiling

An electrochemical biosensor has been developed for sensitive determination of protein kinase activity and inhibition, taking advantage of specific binding of the phosphate groups with TiO2 nanoparticles, on which silver nanoparticles are photo-catalytically deposited to achieve signal enhancement.

Kinome Profiling in Acute Myeloid Leukemia as a New Approach for Target Discovery.

Acute myeloid leukemia (AML) is a heterogeneous disease, characterized by a multitude of genetic events. Activating mutations in receptor tyrosine kinases have been identified in 50% of AML primary blasts, and deregulation of one or more signal transduction pathways including the JAK/STAT, RAS/Raf/MEK/ERK, and PI3K/AKT pathways is common (Kornblau et al., 2006). High-throughput procedures which generate comprehensive descriptions of cellular signaling without a priori assumptions in each sample, would enable us to directly assess a broader range of targets for future treatment strategies. In the present study we identified kinase activity profiles in 22 pediatric leukemia samples (6 acute myeloid leukemia, 9 acute lymphoid leukemia, 3 Philadelphia positive acute lymphoid leukemia and 5 chronic myeloid leukemia) and in various normal tissues such as colon and kidney. Peptide phosphorylation profiles were determined using the Pamchip® tyrosine kinase micro array system (Pamgene International B.V., 's Hertogenbosch, the Netherlands). This array consists of 144 peptides representing key phosphorylation sites of proteins known to be involved in signal transduction processes. We generated a comprehensive description of the phosphotyrosine proteome of all patient samples. Within the variety of profiles in the various leukemia samples, peptide corresponding with phosphorylation consensus sequences for MAP kinases showed remarkable high levels of phosphorylation, whereas in various normal tissue types substantial lower activity was observed on these substrates. These results imply activated MAPK signaling to be a prominent characteristic of leukemia as already described (Towatari et al., 1997). A differential phosphorylation pattern was seen for the RON peptide (macrophage stimulating 1 receptor (c-met-related tyrosine kinase)), only phosphorylated in one of the AML samples, 4 out of 5 CML samples and one Ph+ ALL sample. According to Phospho-ELM and the literature (Follenzi et al., 2000) this tyrosine kinase residue can be phosphorylated via autophosphorylation by RON kinase or transphosphorylation by MET kinase (met proto-oncogene (hepatocyte growth factor receptor)).To verify the role of MET kinase in AML, a cell survival assay was performed with the selective MET kinase inhibitor PHA 665752. Dose dependent decrease in cell survival was observed in three primary AML samples tested with LC50 concentrations ranging from 2 μM to 5 μM. In conclusion, this study describes a new high throughput technique to generate tyrosine phospho-proteomes or even kinome profiles of various leukemic samples in the future. With this technique we found MET to be a new therapeutic target in AML, and it demonstrates the importance of MAP kinase signaling in various leukemic samples. In the era of a rapidly increasing number of small molecule inhibitors this technique will enable us to rapidly identify new potential targets in different kinds of leukemia.

Comparative Phosphoproteomics of Zebrafish Fyn/Yes Morpholino Knockdown Embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

Epidermal growth factor receptor‐dependent PI3K‐activation promotes restitution of wounded human gastric epithelial monolayers

Restitution is a crucial event during the healing of superficial injury of the gastric mucosa involving epithelial cell sheet movement into the damaged area. We demonstrated that growth factors promote the restitution of human gastric epithelial cells. However, the intracellular signaling pathways that transmit extracellular cues as well as regulate basal and growth factor-stimulated gastric epithelial cell migration are still unclear. Herein, confluent human gastric epithelial cell monolayers (HGE-17) or primary cultures of gastric epithelial cells were wounded with a razor blade and the migration response was analyzed in presence or absence of TGFalpha or of pharmacological inhibitors of signaling proteins. Kinase activation profile analysis and phase-contrast microscopy were also performed in parallel. We report that ERK1/2 and Akt activities are rapidly stimulated following wounding of HGE-17 cells. Treatment of confluent HGE-17 cells or primary cultures of gastric epithelial cells with the phosphatidylinositol 3-kinase inhibitor LY294002, but not the MEK1 inhibitor, PD98059, significantly inhibits basal and TGFalpha-induced migration following wounding. Conversely, treatment of wounded HGE-17 cells with phosphatidylinositol(3,4,5)-triphosphate is sufficient to stimulate basal cell migration by 235%. In addition, pp60c-src kinase activity and tyrosine phosphorylation of epidermal growth factor receptors (EGFR) are also rapidly enhanced after wounding and pharmacological inhibition of both these activities strongly attenuates basal and TGFalpha-induced migration as well as Akt phosphorylation levels. In conclusion, the present results indicate that EGFR-dependent PI3K activation promotes restitution of wounded human gastric epithelial monolayers.

Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage

Regulation of protein kinase activities is crucial in both physiology and disease, but analysis is hampered by the multitude and complexity of kinase networks. We used novel peptide array chips containing 1,152 known kinase substrate sequences to profile different kinase activities in renal lysates from homozygous Ren2 rats, a model characterized by hypertension and angiotensin II (ANG II)-mediated renal fibrosis, compared with Sprague-Dawley (SD) control rats and Ren2 rats treated with an angiotensin-converting enzyme inhibitor (ACEi). Five-wk-old homozygous Ren2 rats were left untreated or treated with the ACEi ramipril (1 mg.kg(-1).day(-1)) for 4 wk; age-matched SD rats served as controls (n = 5 each). Peptide array chips were incubated with renal cortical lysates in the presence of radioactively labeled ATP. Radioactivity incorporated into the substrate motifs was measured to quantify kinase activity. A number of kinases with modulated activities, which might contribute to renal damage, were validated by Western blotting, immunoprecipitation, and immunohistochemistry. Relevant kinases identified by the peptide array and confirmed using conventional techniques included p38 MAP kinase and PDGF receptor-beta, which were increased in Ren2 and reversed by ACEi. Furthermore, insulin receptor signaling was reduced in Ren2 compared with control rats, and G protein-coupled receptor kinase (GRK) activity decreased in Ren2 + ACEi compared with untreated Ren2 rats. Array-based profiling of tissue kinase activities in ANG II-mediated renal damage provides a powerful tool for identification of relevant kinase pathways in vivo and may lead to novel strategies for therapy.

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3beta, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 microM in the presence of 0.10 mM ATP.

キナーゼプロファイリングのための新しいリン酸アフィニティー電気泳動法

We developed a novel type of phosphate-affinity polyacrylamide gel electrophoresis using an alkoxide-bridged dinuclear manganese(II) complex as a phosphate-binding tag, Phos-tag. The phosphate-affinity site is a polyacrylamide-bound Mn2+-Phos-tag that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart in an SDS-PAGE gel and the quantitative analysis of kinase reactions. Herein, we describe three applications of protein kinase profiling using the phosphate-affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE). The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein status. The activity profiles of six kinds of kinases were determined using a substrate, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time-course changes in the EGF-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting analysis. The final application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl, was determined using the substrate Abltide-GST and the approved inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the monophosphorylated and nonphosphorylated Abltide-GST bands.

Identification of a 115 kDa MAP-kinase activated by freezing and anoxic stresses in the marine periwinkle, Littorina littorea

The mitogen-activated protein kinase (MAPK) cascade regulates changes in gene transcription by transmitting extracellular stimuli from the plasma membrane to the cell nucleus and has an important role to play in organismal responses to environmental stresses. The activities of MAPKs were investigated in the marine gastropod mollusk, Littorina littorea, a species that tolerates both extracellular freezing and long term oxygen deprivation. In-gel kinase assays revealed the presence of two MAPKs in foot muscle and hepatopancreas, a 42 and a 115 kDa protein. Immunoblot analysis showed that both were MAPK proteins and that one was the periwinkle homologue of p42 ERK2. Size exclusion chromatography confirmed the 115 kDa size of the novel snail MAPK and its role as the dominant MAPK activity in foot muscle. In-gel kinase assays, immunoblotting with phospho-specific ERK antibody, as well as kinase activity profiles from hydroxyapatite chromatography demonstrated that p115 MAPK kinase activity was increased in foot muscle in response to in vivo freezing or anoxia exposures. The results suggest a role for this novel kinase in environmental stress response.