Stable Isotope Labeling With Amino Acids In Cell Culture Research Articles

Comparison of Three Quantitative Phosphoproteomic Strategies to Study Receptor Tyrosine Kinase Signaling

There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.

QUICK identification and SPR validation of signal transducers and activators of transcription 3 (Stat3) interacting proteins

Signal transducers and activators of transcription 3 (Stat3) has been reported to be involved in the pathogenesis of various human diseases and is constitutively active in human multiple myeloma (MM) U266 cells. The Stat3-regulated mechanisms involved in these processes, however, are not fully defined. To further understand the regulation of Stat3 activity, we performed a systematic proteomic analysis of Stat3 interacting proteins in U266 cells. This analysis, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture (SILAC), immunoprecipitation, and quantitative MS. As a result, quantitative mass spectrometry analysis allowed us to distinguish specific Stat3 interacting proteins from background proteins and led to the identification of a total of 38 proteins. Three Stat3 interacting proteins - 14-3-3ζ, PRKCB and Hsp90 - were further confirmed by reciprocal co-immunoprecipitations and surface plasmon resonance (SPR) analysis. Our results therefore not only uncover a number of Stat3 interacting proteins that possess a variety of cellular functions, but also provide new insight into the mechanisms that regulate Stat3 activity and function in MM cells.

Quantitative Phosphoproteomics of CXCL12 (SDF-1) Signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation.

Stable-isotope labeling with amino acids in nematodes.

We describe an approach for the accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted Stable Isotope Labeling with Amino acids in Cell culture (SILAC) for nematodes, by feeding worms a heavy lysine- and arginine-labeled E. coli strain. We also report a genetic solution to remove the arginine-to-proline conversion problem. Combining our approach with quantitative proteomics methods, we characterized the heatshock response in worms.

Quantitative Proteomic Analysis of Cellular Protein Modulation upon Inhibition of the NEDD8-Activating Enzyme by MLN4924

Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.

Quantitative phosphoproteomics of transforming growth factor‐β signaling in colon cancer cells

The transforming growth factor-β (TGF-β) signaling pathway progresses through a series of protein phosphorylation regulated steps. Smad4 is a key mediator of the classical TGF-β signaling pathway; however, reports suggest that TGF-β can activate other cellular pathways independent of Smad4. By investigating the TGF-β-regulated phosphoproteome, we aimed to uncover new functions controlled by TGF-β. We applied titanium dioxide to enrich phosphopeptides from stable isotope labeling with amino acids in cell culture (SILAC)-labeled SW480 cells stably expressing Smad4 and profiled them by mass spectrometry. TGF-β stimulation for 30 min resulted in the induction of 17 phosphopeptides and the repression of 8 from a total of 149 unique phosphopeptides. Proteins previously not known to be phosphorylated by TGF-β including programmed cell death protein 4, nuclear ubiquitous casein and cyclin-dependent kinases substrate, hepatoma-derived growth factor and cell division kinases amongst others were induced following TGF-β stimulation, while the phosphorylation of TRAF2 and NCK-interacting protein kinase are examples of proteins whose phosphorylation status was repressed. This phosphoproteomic screen has identified new TGF-β-modulated phosphorylation responses in colon carcinoma cells.

The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization

Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface.

Large-scale phosphosite quantification in tissues by a spike-in SILAC method

Despite progress in mass spectrometry (MS)-based phosphoproteomics, large-scale in vivo analyses remain challenging. Here we report a 'spike-in' stable-isotope labeling with amino acids in cell culture (SILAC) methodology using standards derived from labeled mouse liver cell lines, using which we analyzed insulin signaling. With this approach we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment, creating a very large, accurately quantified in vivo phosphoproteome dataset.

Metabolic Labeling of Chondrocytes for the Quantitative Analysis of the Interleukin-1-beta-mediated Modulation of Their Intracellular and Extracellular Proteomes

Chondrocytes are widely used as an in vitro model of cartilage diseases such as osteoarthritis (OA). As the unique residents of mature cartilage, they are responsible of the synthesis and release of proteins essential for a proper tissue turnover. In this work, the stable isotope labeling with amino acids in cell culture (SILAC) technique has been standardized in primary human articular chondrocytes (HACs) for quantitative proteomic analyses. Then, it has been employed to study those protein modifications caused by the proinflammatory cytokine Interleukin-1beta (IL-1β), a well-known OA mediator, in these cells. Quantitative analysis of the IL-1β-treated HACs proteome revealed a global increase in cellular chaperones concurrent with a down-regulation of the actin cytoskeleton. HACs secretome analysis led to the identification and quantification of 115 proteins and unveiled the effects of the cytokine on the cartilage extracellular matrix metabolism. Among those modulated proteins, three protein clusters were found to be remarkably increased by IL-1β: proinflammatory mediators and proteases, type VI collagen and proteins known to bind this molecule, and proteins related with the TGF-beta pathway. On the other hand, secretion of aggrecan, two vitamin K-dependent proteins, and thrombospondin, among others, was strongly reduced. Altogether, these data demonstrate the usefulness of metabolic labeling for quantitative proteomics studies in HACs, show the complementarity of intracellular proteome and secretome analyses, and provide a comprehensive study of the IL-1β-mediated effects on these cells. Proteins identified in the secretome approach have a potential use as biomarkers or therapeutic targets for OA.

In Vivo Termini Amino Acid Labeling for Quantitative Proteomics

Quantitative proteomics is one of the research hotspots in the proteomics field and presently maturing rapidly into an important branch. The two most typical quantitative methods, stable isotope labeling with amino acids in cell culture (SILAC) and isobaric tags for relative and absolute quantification (iTRAQ), have been widely and effectively applied in solving various biological and medical problems. Here, we describe a novel quantitative strategy, termed "IVTAL", for in vivo termini amino acid labeling, which combines some advantages of the two methods above. The core of this strategy is a set of heavy amino acid (13)C(6)-arginine and (13)C(6)-lysine and specific endoproteinase Lys-N and Arg-C that yield some labeled isobaric peptides by cell culture and enzymatic digestion, which are indistinguishable in the MS scan but exhibit multiple MS/MS reporter b, y ion pairs in a full mass range that support quantitation. Relative quantification of cell states can be achieved by calculating the intensity ratio of the corresponding reporter b, y ions in the MS/MS scan. The experimental analysis for various proportions of mixed HeLa cell samples indicated that the novel strategy showed an abundance of reliable quantitative information, a high sensitivity, and a good dynamic range of nearly 2 orders of magnitude. IVTAL, as a highly accurate and reliable quantitative proteomic approach, is expected to be compatible with any cell culture system and to be especially effective for the analysis of multiple post-translational modificational sites in one peptide.

Stable Isotope Labeling with Amino Acids in Cell Culture Based Mass Spectrometry Approach to Detect Transient Protein Interactions Using Substrate Trapping

The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the "bait" protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.

Quantitative Proteomic and Interaction Network Analysis of Cisplatin Resistance in HeLa Cells

Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets.

Tumor microenvironment derived biomarkers for the early detection of pancreatic cancer (PDAC).

Abstract e14591 Background: The important role played by activated pancreatic stellate cells (aPSCs) in PDAC has only been recently appreciated. By shaping the tumor microenvironment, APSCs contribute to tumor growth, metastasis and drug resistance. Innovative stable isotope labeling with amino acids in cell culture (SILAC) and multiple reaction monitoring (MRM)-MS provide tools for interrogating human serum samples for aPSC derived biomarkers of PDAC. 600 proteins secreted by aPSCs were previously characterized (Wehr, 2010). Selected proteins were shown to be overexpressed in pancreatic tumors. An immortalized PSC cell line was SILAC labeled; secreted proteins were collected from activated cells and used as labeled internal standards. An MRM-MS method was developed to quantitate the 40 most abundant secreted aPSC proteins in serum. Internal standard proteins were added to human serum samples prior to immunoaffinity (IA) removal of abundant serum proteins, trypsin digested and analyzed by liquid chromatography (LC)/MRM-MS. Powered primary endpoint: characterize the sensitivity and specificity of biomarker panel in a case control study of 10 patients with PDAC and 10 healthy controls. Secondary endpoint: biological pathway analysis of discriminating biomarkers. 183 unique peptides were identified for the selected 40 aPSC proteins. 69 peptides, representing 36 proteins, were reproducibly detected by MS. During method development, a prototypical biomarker candidate, MMP2, was monitored; Western blot and MS analysis showed that MMP2 was not lost by IA processing, and removal of abundant proteins was required for detection of MMP2 by MS. The method was found to be linear over a range of 4-100 fmol/µL media, and met precision criteria of

Deep and Highly Sensitive Proteome Coverage by LC-MS/MS Without Prefractionation

In-depth MS-based proteomics has necessitated fractionation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogenesis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795–806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.

Abstract 1168: Integrated genomic, microRNA (miRNA) and proteomic profiling by stable isotope labeling with amino acids in cell culture (SILAC) of ovarian carcinoma for biomarker discovery

Abstract Ovarian cancer (OCa) is the fifth leading cause of cancer-related deaths in North American women, and the first for gynecologic malignancies. The long-term effectiveness of standard therapy is poor. Thus, there is a need for developing markers for diagnosis, prognosis, and for predicting therapeutic response. Transformation, malignancy and therapy resistance are consequences of changes at the DNA, RNA and protein levels, requiring an integrative approach for biomarker discovery. We, and others, have previously demonstrated that elevated protein levels of Kallikrein 6 (KLK6) in OCa are clinically relevant; and that copy-number gains of the KLK locus (19q13.3/13.4) is associated with elevated levels of KLKs, increasing grade and genomic instability. Recent work has indicated KLK6 protein expression is also regulated by microRNAs (miRNAs). Our profiling of OCa cell lines and primary tumours showed the differential expression of miRNAs, consistent with other published studies. Moreover, miRNAs predicted to target KLK6 were shown to be decreased in a KLK6-overexpressing OCa cell line (OVCAR-3), in comparison to a KLK6-non-expressing cell line (TOV21G) or to miRNAs derived from normal ovarian tissue. Among these are members of the hsa-let-7 family of miRNAs, found also to be in regions of copy-number loss in OVCAR-3. Since miRNAs can affect the protein expression of many genes, the identification of differentially expressed proteins, in addition to KLK6 not only suggests putative biomarkers, but may help elucidate pathways for therapeutic interventions. To identify differentially expressed proteins upon the transient transfection of hsa-let-7 family members into the OVCAR-3 cell line, we utilized Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) coupled to mass spectrometry. OVCAR-3 cultures were labeled separately in light-Arg/Lys and heavy-Arg/Lys isotopes, such that “light” and “heavy” peptides of the same proteins will generate spectra that are different due to a mass shift, thus enabling relative quantification. In control experiments, equal Light/OVCAR3 and Heavy/OVCAR3 total protein mixtures were profiled with 2,800 proteins identified, and 2,465 quantified. Over 94% of these quantified proteins showed a heavy:light ratio between 0.8 and 1.2, making this a robust system for distinguishing differentially expressed proteins. Light/OVCAR-3 was transfected with hsa-let-7a or hsa-let-7e while heavy/OVCAR-3 was transfected with a scrambled miRNA control. KLK6-specific ELISA confirmed its decrease by 50% in transfected cultures over controls. Preliminary SILAC profiling has identified a number of differentially expressed proteins upon transfection with the miRNAs, which will be discussed in the context of OCa pathogenesis and implications for novel biomarker panels and proteomic signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1168. doi:10.1158/1538-7445.AM2011-1168

Abstract 4870: High-throughput proteomics using stable isotope labelling with amino acids in cell culture (SILAC) reveals potential modulators of androgen-independent prostate cancers

Abstract Prostate cancer is the most common malignancy and the second leading cause of cancer related deaths in men. One common treatment is androgen-deprivation therapy, which reduces symptoms in most patients. However, over time patients develop tumours that are androgen-independent and ultimately fatal. The mechanisms that cause this transition remain largely unknown, and as a result, there are no effective treatments against androgen-independent prostate cancer. As a model for androgen-independence, we used the LNCaP cell line which is naturally androgen-dependent grown in androgen depleted conditions for a 6 month period to generate the androgen-independent cell line LNCaP-SF. Preliminary experiments showed that the LNCaP-SF cell line had higher proliferation/viability rates in androgen-depleted conditions, and had increased expression of the androgen receptor protein, a key modulator in prostate cancer progression. Our next goal was to assess to differential global protein expression as well as differential expression of secreted proteins from each of the two cell lines. To identify differentially expressed proteins, we utilized Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) coupled to mass spectrometry. LNCaP cells were labelled in growth media containing light-Arg/Lys whereas LNCaP-SF was labelled in heavy Arg-Lys media. After proteomic analysis, we were able to quantify 3355 proteins with at least one peptide hit. From these proteins, using stringent criteria, 99 and 82 proteins were found to be up-regulated and down-regulated respectively, in LNCaP-SF cells compared to LNCaP cells. Using various bioinformatics tool, we found that genes involved in fatty acid degradation and ketogenesis as important pathways that with over-expressed in LNCaP-SF cells. We performed qPCR validation of genes involved in each of these pathways and found that LNCaP-SF cells generally had an increase in the expression of many these genes, compared to LNCaP cells. These preliminary results show that genes involved in fatty acid degradation and ketogenesis pathways could serve as potential biomarkers for androgen-independent prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4870. doi:10.1158/1538-7445.AM2011-4870

Abstract 3131: A novel tyrosine kinase signaling pathway in human basal breast cancer

Abstract Basal-type breast cancers account for up to 25 % of breast cancer cases, depending on the patient population. These cancers usually lack estrogen, progesterone and erbB2 receptors and are therefore resistant to endocrine or trastuzumab therapy, and markers that stratify this subset according to clinical behaviour have yet to be identified. Consequently, basal breast cancers present a major treatment dilemma. In order to identify potential therapeutic targets and prognostic markers for this disease subgroup, we are currently undertaking a detailed characterization of tyrosine kinase signaling networks in basal breast cancer cells. Mass spectrometry (MS)-based global profiling of the tyrosine phosphoproteome of basal breast cancer cells identified a prominent Src family kinase (SFK) signaling network that featured high expression of the SFK Lyn (Hochgräfe et al, Cancer Res 2010 doi: 10.1158/0008-5472.CAN-10-0911). In order to identify Lyn substrates, we used stable isotope labelling with amino acids in cell culture (SILAC) in combination with immunoaffinity-based phosphotyrosine enrichment and LC-MS/MS to profile cellular tyrosine phosphorylation following transient siRNA-mediated Lyn knockdown in MDA-MB-231 and BT-549 basal breast cancer cells. This identified several Lyn substrates characteristic of both cell lines, which included annexin A2 (Y42), 40S ribosomal protein S10 (Y12) and SgK269 (Y635). The latter is a relatively understudied kinase that contains several tyrosine phosphorylation sites that are likely to act as recruitment sites for SH2/PTB domain-containing signalling proteins, and a C-terminal kinase-like domain that lacks the consensus DFG motif usually regarded as essential for kinase activity. Protein expression of SgK269 was markedly higher in basal, versus luminal, breast cancer cells, while mRNA transcript levels in the two cell types were similar, indicating post-transcriptional regulation of SgK269 expression. Phosphorylation of SgK269 on Y635 could be detected readily in basal breast cancer cells. Overexpression of SgK269 in MCF-10A immortalized mammary epithelial cells led to a more elongated morphology in monolayer and perturbed acinar morphogenesis in Matrigel, resulting in large, multilobular acini that failed to undergo proliferative suppression in long-term culture. SgK269-overexpressing cells also exhibited enhanced tyrosine phosphorylation of Stat3. In addition, we undertook stable shRNA-mediated knockdown of SgK269 in BT-549 cells. This reduced soft agar growth and Stat3 activation. These studies identify a novel oncogenic signalling pathway in basal breast cancer involving the kinases Lyn and SgK269, which may represent a target for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2011-3131

Abstract 578: Roles of mitochondrial protein kinases in targeted cancer therapy

Abstract Targeted cancer therapy is based on the curious dependence that some cancer cells have on a single oncogenic molecule, typically a protein kinase (the phenomenon of “oncogene addiction”). However, the molecular mechanisms of such dependence are incompletely understood. We have adopted a quantitative proteomic approach to investigate the phenomenon that MET-overexpressing cancer cells are paradoxically more sensitive to MET inhibition than cells with basal MET expression. Unexpectedly, we found that mitochondria are a direct target of MET kinase inhibition, in addition to inhibition of plasma membrane MET. Effects on activated MET in the mitochondria of cancer cells that are sensitive to MET inhibition could reflect a novel and critical non-canonical mechanism that contributes to the efficacy of MET-targeted therapeutics. We hypothesize that protein kinases which aberrantly translocate to mitochondria modulate mitochondrial proteins and functions. Here we have employed advanced LC-MS/MS-based stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomic approaches to identify, quantify and functionally annotate phosphorylated mitochondrial proteins of various cancer cell lines, including those that are sensitive or resistant to targeted agents. We identified several phosphorylated protein kinases e.g. MET, EGFR, HER2 and HER3, that were translocated to mitochondria of cancer cells. In addition to documenting the first kinome and phosphoproteome of mitochondria in cancer cells, we present evidence showing that the aberrant presence of protein kinases in mitochondria predicts sensitivity to specific kinase inhibitors. In conclusion, our data reveal a novel mitochondrial mechanism of oncogene addiction, and suggest that mitochondrial protein kinases might play decisive roles in molecularly-directed therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2011-578

Study of Neurotrophin-3 Signaling in Primary Cultured Neurons using Multiplex Stable Isotope Labeling with Amino Acids in Cell Culture

Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles.

Protein expression profiles of C3H 10T1/2 murine fibroblasts and of isogenic cells transformed by the H1047R mutant of phosphoinositide 3-kinase (PI3K)

We have used stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with tandem mass spectrometry to characterize the proteomes of two isogenic cell lines that differ in the expression of a single oncoprotein,p110α of PI3K, carrying the H1047R mutation. 51,510 peptides were identified and assigned to 4,201 proteins. Most notable among the proteins that show increased expression in the oncogenically transformed cells are several involved in the interferon response including Isg15, Ifit1, Igtp and Oas2 (interferon stimulated gene 15, interferon-induced protein with tetratricopeptide repeats 1, interferon gamma-inducible GTP-binding protein, 2'-5'-oligoadenylate synthetase 2). Prominent among the downregulated proteins are several involved in cell adhesion as well as proteins that are affected by the negative feedback from PI3K signaling. The differential expressions documented in this analysis suggest novel links between oncogenic PI3K and several signaling pathways. These links will be explored in future studies.