OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3′ kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non–receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.

Tyrosine phosphorylation and dephosphorylation play important roles in the regulation of normal and neoplastic cell growth, attachment, and survival. Receptor tyrosine kinases, such as Kit, are known to generate strong growth and survival signals once activated, and inhibition of such signals is proposed to result in reduced cell proliferation and increased apoptosis. There is considerable evidence that expression of mutant alleles encoding constitutively active Kit receptor molecules is a major factor driving tumor growth in both mast cell leukemias/mastocytosis and gastrointestinal stromal tumors (GIST; refs. 1, 2). The most prevalent Kit mutations in GIST are within the regulatory juxtamembrane domain (exon 11, 67% of GIST patients), although a small percentage of GIST patients express activating mutations within the extracellular portion or kinase domain of Kit, or mutant forms of the closely related receptor tyrosine kinase platelet-derived growth factor receptor α (PDGFRα; ref. 3) The presence of Kit mutations has been correlated with poorer prognosis in GIST (4, 5); germ line inheritance of such mutations has been found to result in marked susceptibility to GIST (6), a phenotype that was also recapitulated in a transgenic mouse model system (7). The benefit of Kit inhibition in GIST has been shown using STI-571 (Gleevec), an inhibitor of PDGFR, Abl, and Kit, resulting in Food and Drug Administration approval of this agent for the treatment of malignant metastatic/nonresectable GIST (810). The human mast cell leukemia line HMC-1 (11) expresses an exon 11 mutant form of Kit (V560G) resembling the most common type of mutant found in GIST patients (12, 13). A variant of the HMC-1 cell line has also been described that expresses an additional kinase domain mutation (D816V; ref. 13), which was not present in the clone used here. The phenotypic response of these cell lines to a selective Kit inhibitor (STI-571) was found to be dependent on the type of mutation present, with the V560G/D816V mutant being insensitive to STI-571, whereas proliferation of the V560G mutant line was potently inhibited by STI-571 (1416), reflecting the different sensitivities of the mutant Kit proteins to kinase inhibition by STI-571 (17). Thus, the cellular phenotype of the V560G mutant HMC-1 line is highly dependent on the kinase activity of the mutant Kit enzyme. Therefore, this cell line represents a useful model system for analysis of the effects of Kit inhibition on cell signaling events and phenotypic characteristics regulated by the activated Kit receptor.

OSI-930 is a potent and selective inhibitor of the closely related receptor tyrosine kinases Kit, KDR, and PDGFRβ (Fig. 1) that exhibits antitumor activity in tumor xenograft models representing a broad range of tumor types. We have used this small-molecule kinase inhibitor to study the temporal consequences of mutant Kit inhibition on mast cell leukemia signaling pathways. The principle aim of the study was to define and measure components of the Kit signaling modulated by kinase inhibition in a model of mast cell leukemia. Kit inhibition and dephosphorylation markedly reduced downstream signaling where phosphorylation and activation of the Ras-Erk, phosphatidyl inositol-3′ (PI-3′) kinase-Akt-S6K, and signal transducers and activators of transcription (STAT) pathways were reduced. A multiplex isobaric labeling method (18) coupled to antiphosphotyrosine affinity chromatography was used to selectively identify and measure proteins phosphorylated or tyrosine or complexed therewith over multiple time points following Kit kinase perturbation. Two hundred and eighty-two proteins were unequivocally identified and their abundance measured 1, 4, and 24 hours after exposure to OSI-930. Time-dependent functional crosstalk between Kit, adapter proteins, tyrosine phosphatases, and focal adhesion components was observed and quantitated. Abundance measurements were confirmed by cell pellet microarray immunohistochemistry, by immunoblot, and/or by isotope-coded affinity tag (19) labeling approaches with good overall correlation between techniques. The combined use of multiplex labeling and protein expression clustering allowed a focus on specific classes of substrates altered temporally in response to kinase inhibition.

Figure 1.

The chemical structure of the thiophene inhibitor (OSI-930) of Kit, KDR, and PDGFR kinases used to inhibit constitutively active mutant Kit kinase function in the mast cell leukemia line HMC-1. IC50 values for Kit, KDR, and PDGFRβ kinases are indicated.

Figure 1.

The chemical structure of the thiophene inhibitor (OSI-930) of Kit, KDR, and PDGFR kinases used to inhibit constitutively active mutant Kit kinase function in the mast cell leukemia line HMC-1. IC50 values for Kit, KDR, and PDGFRβ kinases are indicated.

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Preparation of Immobilized Antibody Affinity Resins

Antiphosphotyrosine immunoaffinity resins were prepared by covalent coupling to solid support as previously described (20), where disuccinimidyl suberate (Pierce, Rockford, IL) was used as the cross-linker. Freshly prepared immunoaffinity resins were used for each biological experiment to maximize binding and reduce carry-over. Briefly, antiphosphotyrosine antibodies PY20 (Exalpha Biologicals, Inc, Watertown, MA) and PY100 (Cell Signaling Technology, Beverly, MA) were mixed in an 5:1 ratio and bound to Protein G resin (Pierce) for 30 minutes at room temperature, followed by cross-linking with 5 mmol/L disuccinimidyl suberate for 1 hour at room temperature and washing with TBS (pH 7.2). Noncovalently bound IgG was removed by rapidly washing with 0.2 mol/L sodium citrate (pH 2.8). Cross-linked antibody resin was then stored at 4°C in TBS until use.

Preparation of HMC-1 Cell Lysate, Antiphosphotyrosine Affinity Chromatography, and Protein Immunodetection

Approximately 2 × 109 HMC-1 cells (V560G with no mutation at D816; data not shown) were grown as spinner cultures at 37°C in IMDM with 10% fetal bovine serum, supplemented with 1% l-glutamine and 1.2 mmol/L α-monothioglycerol. The Kit receptor kinase inhibitor OSI-930 (Fig. 1; 0.5 μmol/L) was added to HMC-1 cells for 0, 1, 4, or 24 hours before lysis. Cells were harvested by centrifugation (1,000 × g, 5 minutes) and washed once with PBS followed by a second wash with ice-cold PBS containing 100 μmol/L sodium orthovanadate before lysis for ∼3 minutes in 50 mmol/L HEPES (pH 7.5) containing 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1 mmol/L AEBSF, 0.8 μmol/L aprotinin, 20 μmol/L leupeptin, 40 μmol/L beestatin, 15 μmol/L pepstatin A, 14 μmol/L E-64 (1:100 dilution of protease inhibitor cocktail P8340; Sigma, St. Louis, MO), sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole (1:100 dilution of phosphatase inhibitor cocktail P5726; Sigma). Insoluble material was removed by centrifugation (13,000 × g, 10 minutes, 4°C) and the protein concentration was determined by micro–bicinchoninic acid assay (Pierce). To reduce nonspecific protein binding to the affinity resin, lysates were precleared by incubation with Protein G resin for 30 minutes at 4°C. Antibody resins were then incubated with HMC-1 cell lysates for ∼5 hours at 4°C with rotation. When loading protein for antiphosphotyrosine affinity chromatography, cell equivalents rather than protein equivalents were used to avoid bias associated with combined kinase inhibition and antiphosphotyrosine selection. Antibody-antigen complexes were washed with >200 volumes of 10 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl at 4 °C, and bound proteins were then eluted with 0.1% trifluoroacetic acid, 5% methanol in water, and were lyophilized and stored at −80°C until further use. Protein immunodetection was done by electrophoretic transfer of SDS-PAGE–separated proteins to nitrocellulose, incubation with antibody, and chemiluminescent detection (PicoWest; Pierce). Antibodies used were as follows: phospho-AktS473 (Cell Signaling), Akt (Cell Signaling), phospho-p44/42 mitogen-activated protein kinase (MAPK)T202/Y204 (Erk1/2; Cell Signaling), phospho-Src familyY416 (Cell Signaling), β-actin (Sigma), phospho-STAT3Y705 (Cell Signaling), phospho-S6S235/236 (Cell Signaling), phospho-KitY721 (Cell Signaling), phospho-KitY703 (BioSource International, Camarillo, CA), Kit (Lab Vision, Fremont, CA), and poly-ADP ribose polymerase (PARP; Cell Signaling).

Peptide Identification by Liquid Chromatography Tandem Mass Spectrometry Fragment Ion Spectra Database Searching

Proteins isolated by antiphosphotyrosine affinity chromatography were denatured in 0.5 mol/L triethylammonium bicarbonate, 0.1% SDS, reduced with 5 mmol/L Tris-(2-carboxyethyl)phosphine at 60°C for 60 minutes; free cysteines reacted with 10 mmol/L methyl methanethiosulfonate at room temperature for 10 minutes and proteolytically cleaved with trypsin (10 μg, 37°C, 16 hours). Peptide amino-terminal α-amino and lysine ε-amino groups were labeled with isobaric tags by NHS ester coupling essentially as described (18) using a different isobaric tag to label peptides from different time points. After labeling, the peptides were further purified by cation exchange chromatography and C18 desalting steps. Strong cation exchange chromatography was done using a 4.6 × 5 mm cation exchange column packed with polysulfoethyl A resin (OptimizeTechnologies, Oregon City, OR). Peptides were desalted before on-line liquid chromatography tandem mass spectrometry (LC-MS/MS) by gradient C18 reverse-phase chromatography (0.5 × 50 mm; Phenomenex, Torrance, CA) in 0.1% trifluoroacetic acid and 4% to 70% acetonitrile over 20 minutes with UV detection at 214 nm.

Peptides were introduced into the quadrapole time-of-flight mass spectrometer by reverse-phase (C18) high-performance liquid chromatography using 0.1 × 150 mm columns (MagicC18, Michrom Bioresources, Auburn, CA), developed using a 2% to 60% acetonitrile, 0.1% formic acid gradient with a flow rate of ∼200 nL/min. The electrospray source was fitted with an uncoated tapered fused silica tip (10 μm ID; New Objective, Cambridge, MA) to which a voltage of ∼2.4 kV was applied. Information-dependent MS and MS/MS acquisitions were made on an orthogonal quadrapole time-of-flight instrument (SCIEX, Toronto, Ontario, Canada) using a 0.8-second survey scan (m/z 400–1,200) followed by three consecutive 2-second product ion scans of 2+, 3+, and 4+ parent ions with a 4-minute exclusion period as previously described (20). Ions were stored in the second quadrapole and released in synchrony with the pulsing of ions in time-of-flight detector. MS data was collected using Analyst QS (Version 1.0 SP8; Applied Biosystems, Inc., Foster City, CA). Proteins were identified from survey and product ion spectra data, with an MS and MS/MS mass tolerance of 0.15 Da, using both SwissProt and Celera databases with the Pro Quant (Applied Biosystems) search program. A comparison of the search algorithm with Mascot and SONAR was previously described (20). One missed tryptic cleavage was allowed and posttranslational modifications considered included cysteine derivitization, STY phosphorylation, deamidation, and oxidation. Pro Quant confidence levels of ≥90% with scores of ≥20 were considered, after which spectra were inspected manually. Peptide assignments to more than one protein were prevented by manual sorting and by use of algorithms within Pro Group Viewer (Applied Biosystems). Peptide expression ratios were averaged to yield a single protein expression value for each time point. Proteins were clustered by temporal log2 protein expression ratios using Euclidian hierarchical methods (UPGMA) and self-organizing maps (Spotfire DecisionSite 8.0).

Preparation of Cell Microarrays and Expression Profiling

Cells were washed with PBS, scraped from the plates, and resuspended in a small volume of PBS. Formalin was added to a final concentration of 10% and the cells were fixed for 30 minutes at room temperature, pelleted by centrifugation, washed twice with PBS, and resuspended in a small volume of melted Histogel (Richard Allen Scientific, Kalamazoo, MI). The mixture was transferred to cloning cylinders and allowed to solidify on ice before overnight fixation in 10% formalin (4°C). Cell pellets were processed and embedded in paraffin per standard procedures. Cell arrays were prepared from paraffin-embedded cell suspensions using a manual arrayer (Beecher Instruments, Sun Prairie, WI) and a core size of 1.0 mm. Cell samples from various time points and concentrations were arrayed together with cell and tissue control samples. Paraffin sections were prepared from the cell arrays, mounted onto glass slides, and stained immunohistochemically with phosphorylation-specific antibodies according to the instructions of the manufacturer (Cell Signaling Technology). The following polyclonal and monoclonal antibodies from Cell Signaling Technology were used for profiling: phospho-S6 ribosomal proteinS235/236; phospho-S6 ribosomal proteinS240/244; phospho-Akt substrate; phospho-AktS473; phospho-p44/42 MAPKT202/Y204; phospho-CrkLY207; phospho-Src FamilyY416; phospho-SrcY527; phospho-Stat3Y705; phospho-Stat6Y641; phosphopaxillinY118; and phospho–c-Cbl (Y774). The antibodies were extensively analyzed on test arrays before cell array profiling to validate specificity and to determine titration points for maximal dynamic range. Expression levels were scored subjectively by comparing the intensity of control and treated samples. Small increases and decreases were scored as +1 or −1, respectively; large changes were scored as +2 or −2; 0 denoted no change.

Cellular Responses to Kit Kinase Inhibition

The mast cell leukemia line HMC-1, which expresses a constitutively active juxtamembrane mutant Kit receptor tyrosine kinase (V560G), was used as a model system in which a large percentage of the total phosphotyrosine-containing proteins are dependent, either directly or indirectly, on the tyrosine kinase activity of the mutant Kit receptor. The thiophene kinase inhibitor OSI-930 (Fig. 1) markedly inhibited the autophosphorylation of Kit within 1 hour of exposure to 500 nmol/L inhibitor on both Y703 and Y721 in HMC-1 cells, with little change in total Kit levels (Fig. 2A). This was accompanied by a marked decrease in the PDK2 phosphorylation of Akt on S473, suggestive of a block to the coupling of Kit to the p85 subunit of PI-3′ kinase. No change in total Akt level was observed. This reduction in Kit autophosphorylation was observed after 2 hours at an OSI-930 concentration of 100 nmol/L (Fig. 2B), where coincident decreases in phospho-S6S235/236 and phospho-ErkT202/Y204 were observed. These data, showing OSI-930–mediated reduction in phospho-S6, phospho-Akt, and phospho-Erk, were confirmed by immunohistochemical staining of HMC-1 formalin-fixed paraffin-embedded cell pellets (Table 1), although the less sensitive immunohistochemical methodology underestimated expression changes at low OSI-930 concentrations. Taken together, these data indicated OSI-930–attenuated downstream signaling through both Ras-Raf-Mek-Erk and PI-3 kinase-Akt-S6K pathways. OSI-930 also reduced, but did not abolish, phosphorylation of Y705 and activation of STAT3 in HMC-1 cells. The reduction in STAT3 phosphorylation associated with Kit kinase inhibition was confirmed by HMC-1 cell pellet immunohistochemistry (Table 1). These data suggested that OSI-930 attenuated the Kit-dependent phosphorylation of STAT3, but other kinases unresponsive to OSI-930 also contributed to STAT3 phosphorylation in HMC-1 cells. Incubation of HMC-1 with OSI-930 (500 nmol/L) for 24 hours caused apoptosis of HMC-1 cells as measured by immunoblots detecting the caspase cleavage products of PARP (Fig. 2C). To better define and measure components of the Kit signaling pathway, tyrosine phosphorylated proteins and complexes were isolated by antiphosphotyrosine affinity selection and identified and quantitated by a novel LC-MS/MS approach.

Figure 2.

A, OSI-930 time-dependent inhibition of Kit autophosphorylation and signaling through the PI-3′ kinase-Akt-S6K pathway was measured. Phosphorylation of Kit on tyrosines Y703 and Y721 and Akt at S473 was undetectable within 1 h of Kit kinase inhibition of HMC-1 cells with 500 nmol/L OSI-930. The phosphorylation of Akt was partially restored after 24 h of compound exposure. B, OSI-930 dose-dependent inhibition of Kit autophosphorylation and signaling through the Ras-Raf-Mek-Erk pathway, the PI-3′ kinase-Akt-S6K pathway, and STAT pathways was measured by immunoblot detection. The phosphorylation of ribosomal S6S235/236, Erk1/2T202/Y204, and mutant KitY721 was undetectable after 2 h of incubation of HMC-1 cells with 100 nmol/L of the Kit/KDR inhibitor OSI-930. The phosphorylation of the transcription factor STAT3Y705 was partially inhibited at 100 nmol/L OSI-930, where inhibition was not increased with higher compound concentration. β-actin served as a protein loading control. C, inhibition of Kit tyrosine kinase activity with 500 nmol/L OSI-930 for 24 h causes apoptosis of HMC-1 cells. Apoptosis was measured by caspase-dependent PARP cleavage measuring the large and small PARP fragments (89 and 24 kDa, respectively) of resulting from proteolytic cleavage. Little or no PARP cleavage was observed with the DMSO solvent control.

Figure 2.

A, OSI-930 time-dependent inhibition of Kit autophosphorylation and signaling through the PI-3′ kinase-Akt-S6K pathway was measured. Phosphorylation of Kit on tyrosines Y703 and Y721 and Akt at S473 was undetectable within 1 h of Kit kinase inhibition of HMC-1 cells with 500 nmol/L OSI-930. The phosphorylation of Akt was partially restored after 24 h of compound exposure. B, OSI-930 dose-dependent inhibition of Kit autophosphorylation and signaling through the Ras-Raf-Mek-Erk pathway, the PI-3′ kinase-Akt-S6K pathway, and STAT pathways was measured by immunoblot detection. The phosphorylation of ribosomal S6S235/236, Erk1/2T202/Y204, and mutant KitY721 was undetectable after 2 h of incubation of HMC-1 cells with 100 nmol/L of the Kit/KDR inhibitor OSI-930. The phosphorylation of the transcription factor STAT3Y705 was partially inhibited at 100 nmol/L OSI-930, where inhibition was not increased with higher compound concentration. β-actin served as a protein loading control. C, inhibition of Kit tyrosine kinase activity with 500 nmol/L OSI-930 for 24 h causes apoptosis of HMC-1 cells. Apoptosis was measured by caspase-dependent PARP cleavage measuring the large and small PARP fragments (89 and 24 kDa, respectively) of resulting from proteolytic cleavage. Little or no PARP cleavage was observed with the DMSO solvent control.

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Table 1.

HMC-1 cells were exposed to OSI-930 (10 μmol/L) for 3 hours before fixation, embedding in semisolid media followed by paraffin

AntibodyOSI-930
0.1 μmol/L1 μmol/L10 μmol/L
Phospho-S6 ribosomal protein (S235/236−2 −2 −2 
Phospho-S6 ribosomal protein (S240/244−2 −2 −2 
Phospho-Akt substrate −2 −2 −2 
Phospho-Akt (S473−1 −1 
Phospho-S6 kinase (T421/424−1 −1 −1 
Phospho-Erk (T202/Y204−1 −2 
Phospho-CrkL (Y207−1 −1 −2 
Phospho-Src family (Y416−1 −2 
Phospho-Stat3 (Y705−1 −2 −2 
Phospho-Stat6 (Y641−1 −1 −2 
Phosphopaxillin (Y118−1 −2 
Phospho–c-Cbl (Y774−1 −1 
p21/WAF1 −1 −1 −1 
AntibodyOSI-930
0.1 μmol/L1 μmol/L10 μmol/L
Phospho-S6 ribosomal protein (S235/236−2 −2 −2 
Phospho-S6 ribosomal protein (S240/244−2 −2 −2 
Phospho-Akt substrate −2 −2 −2 
Phospho-Akt (S473−1 −1 
Phospho-S6 kinase (T421/424−1 −1 −1 
Phospho-Erk (T202/Y204−1 −2 
Phospho-CrkL (Y207−1 −1 −2 
Phospho-Src family (Y416−1 −2 
Phospho-Stat3 (Y705−1 −2 −2 
Phospho-Stat6 (Y641−1 −1 −2 
Phosphopaxillin (Y118−1 −2 
Phospho–c-Cbl (Y774−1 −1 
p21/WAF1 −1 −1 −1 

NOTE: Cell pellet cores (1 mm in diameter) were arrayed onto glass slides and stained with the indicated antibodies. Scores were as follows: −2, strong reduction; −1, reduction; 0, no change; 1, increase; 2, strong increase.

Quantitation of Temporal Changes in Cellular Tyrosine Phosphorylation following Inhibition of Mutant, Constitutively Active Kit in HMC-1 Cells

In HMC-1 cells, the stem-cell factor receptor Kit was the predominant phosphoprotein detected by antiphosphotyrosine immunoblot (data not shown). Consistent with these data, Kit showed the greatest peptide coverage by LC-MS/MS and Kit represented a major scaffolding protein by which associated proteins and phosphoproteins were enriched. In typical immunoblot or proteomic analyses of cell signaling pathways, fixed analytes or time points are examined in a given experiment. Here, we examined the time-dependent cellular changes associated with inhibition of the Kit receptor tyrosine kinase by isolation and quantitation of phosphotyrosine-containing proteins and complexes dependent on phosphotyrosine for their assembly. A novel multiplex isobaric labeling approach allowed protein quantitation at multiple time points within the same experiment (Fig. 3A). Antibody capture methods can suffer from an unacceptable level of nonspecific binding, confounding the identification of proteins specifically interacting with a given target. The use of a Kit kinase inhibitor allowed us to discern pharmacologically regulated events, relatively insensitive to the effects of nonspecific binding. Several approaches to minimize nonspecific binding previously described (20) were also used. Peptides derived from affinity-selected proteins were modified with isobaric labels that react with the free α and ε amino groups of amino termini and lysine groups (Fig. 3A). Four different labels were used to distinguish samples from the four time points analyzed in these experiments, each label having essentially the same mass, but differing in the size of the diagnostic fragment ions (m/z 114, 115, 116, 117) that are released on collision-induced dissociation (18) within a quadrapole time-of-flight mass spectrometer. The area of these mass peaks provide a measure of quantitation of the peptide (and hence the protein) under the biological and isolation conditions used, in this case inhibition of protein tyrosine phosphorylation brought about by pharmacologic inhibition of Kit tyrosine kinase activity. Because the labeled peptide samples are pooled and subjected to nano–LC-MS/MS within a single experiment, the four isobaric-labeled peptides show identical retention times by high-performance liquid chromatography and, with isobaric masses, are coselected for fragment ion generation, thereby eliminating variations in ion suppression between individual labeled peptides. Quantitation was achieved by the release of peptide tags during collision-induced dissociation with the mass spectrometer, in a region of MS/MS peptide fragment ion spectra with relatively low noise and high dynamic range. The multiplex isobaric approach, using pooled peptides, has great advantage when attempting to measure peptide abundance between experimental conditions or between replicate experiments.

Figure 3.

A, isobaric peptide labeling strategy used for protein identification and peptide quantitation. Control and treatment proteins from control and 1-, 4-, and 24-h OSI-930–treated HMC-1 cells were isolated by detergent extraction and antiphosphotyrosine affinity selection. Proteins were proteolytically cleaved with trypsin and derivatized with amino-reactive isobaric tags for relative and absolute quantitation. The samples were combined and labeled peptides were purified by batch strong cation exchange and reverse-phase liquid chromatography (C18). Peptides were analyzed by LC-MS/MS using nano–reverse-phase fractionation and information-dependent MS to MS/MS switching. Proteins were identified from peptide fragment ion spectra and computer searching of the SwissProt and Celera databases using Pro Quant software. The isobaric tags fragment to yield ions at 114 m/z (control), 115 m/z (1 h Kit kinase inhibition), 116 m/z (4 h inhibition), and 117 m/z (24 h inhibition). Ratios between tag peak areas for control and treatment samples allowed for quantitation of the temporal effects of Kit inhibition for a given peptide. Peptide ratios for a given protein were averaged to provide a treatment ratio and statistic associated with Kit inhibition. B and C, direct quantitation of temporal changes in Kit (B) and Shp-1 (C) phosphorylation associated with inhibition of Kit kinase activity, measured by isobaric labeling and tag release by LC-MS/MS fragmentation. Fragment ion spectra for Kit (Y703) and Shp-1 (Y566) phosphopeptides are shown. The quantitation tags (m/z 114, 115, 116, and 117) were released by collision-induced dissociation within an orthogonal quadrapole time-of-flight mass spectrometer. The area under the curve of the four tag peaks reflects the abundance of the parent protein at the four time points [control (0), 1, 4, and 24 h].

Figure 3.

A, isobaric peptide labeling strategy used for protein identification and peptide quantitation. Control and treatment proteins from control and 1-, 4-, and 24-h OSI-930–treated HMC-1 cells were isolated by detergent extraction and antiphosphotyrosine affinity selection. Proteins were proteolytically cleaved with trypsin and derivatized with amino-reactive isobaric tags for relative and absolute quantitation. The samples were combined and labeled peptides were purified by batch strong cation exchange and reverse-phase liquid chromatography (C18). Peptides were analyzed by LC-MS/MS using nano–reverse-phase fractionation and information-dependent MS to MS/MS switching. Proteins were identified from peptide fragment ion spectra and computer searching of the SwissProt and Celera databases using Pro Quant software. The isobaric tags fragment to yield ions at 114 m/z (control), 115 m/z (1 h Kit kinase inhibition), 116 m/z (4 h inhibition), and 117 m/z (24 h inhibition). Ratios between tag peak areas for control and treatment samples allowed for quantitation of the temporal effects of Kit inhibition for a given peptide. Peptide ratios for a given protein were averaged to provide a treatment ratio and statistic associated with Kit inhibition. B and C, direct quantitation of temporal changes in Kit (B) and Shp-1 (C) phosphorylation associated with inhibition of Kit kinase activity, measured by isobaric labeling and tag release by LC-MS/MS fragmentation. Fragment ion spectra for Kit (Y703) and Shp-1 (Y566) phosphopeptides are shown. The quantitation tags (m/z 114, 115, 116, and 117) were released by collision-induced dissociation within an orthogonal quadrapole time-of-flight mass spectrometer. The area under the curve of the four tag peaks reflects the abundance of the parent protein at the four time points [control (0), 1, 4, and 24 h].

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Proteins from equivalent cell populations were isolated by phosphotyrosine capture under control conditions and after 1, 4, and 24 hours of Kit kinase inhibition. Multiple biological and LC-MS/MS experiments were done for both protein identification and for peptide quantitation. Several statistics were generated. For proteins identified with two or more peptides, 1,041 unique peptides with confidence ≥90% and scores of ≥20 were assigned by searching of both Swissprot and Celera protein databases. The mean percentage deviation of Kit peptide expression ratios was 24%. Two hundred and eighty-two proteins defined by two or more peptides were identified where the mean confidence of peptides supporting protein identification (Table 2) was 98.1% (±0.8) with a mean score of 29.5 (±2.7). The complete data set is provided as Supplementary Table S1.5

5

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Eight proteins were identified through manual confirmation of multiple spectra of a single unique peptide. Protein identification approaches were conformed to the guidelines of Baldwin (21) and Carr et al. (22). The mean expression ratios between Kit inhibitor treatment and control samples were measured by determining ratios of peak areas for the m/z 114 (control), 115 (1 hour), 116 (4 hours), and 117 (24 hours) across all peptides for a given protein. The mean log2 protein expression ratios after 1 and 4 hours of Kit kinase inhibition were −0.64 and −0.66, respectively. The attenuation of Kit autophosphorylation in the antiphosphotyrosine fraction was apparent at these time points (Fig. 2A). The data indicated that whereas the majority of proteins were unchanged after 1 or 4 hours of inhibition, a significant number of proteins were down-regulated in the antiphosphotyrosine fraction in response to compound action. A marked loss of overall signal was observed by 24 hours, with a mean log2 expression ratio of −1.01. This correlated with the onset of apoptosis that resulted from attenuation of mutant Kit signaling in HMC-1 cells, because significant cleavage of PARP was observed at the 24-hour time point (Fig. 2B).

Table 2.

Protein expression clusters identified by antiphosphotyrosine selection from HMC-1 cells exposed to the Kit kinase inhibitor OSI-930 (0.5 μmol/L) for 0, 1, 4, or 24 hours

AccProtein namePep%CScDst1 hP14 hP424 hP24ClSOM
P10721 Kit (mast/stem cell growth factor receptor precursor) 29 98 32 17 −3.57 0.00 −3.93 0.00 −3.81 0.00 11 
P27361 MAPK 3 (Erk1) 98 29 15 −2.99 0.03 −2.51 0.05 −3.35 0.02 11 
P28482 MAPK 1 (Erk2) 97 28 14 −2.71 0.00 −1.83 0.16 −2.92 0.00 11 
P62993 Grb2 (adapter protein) 99 33 19 −3.03 0.00 −0.95 0.00 −2.64 0.00 11 
P22681 CBL E3 ubiquitin protein ligase 98 28 14 −1.55 0.00 −1.46 0.01 −2.63 0.00 11 
P62988 Ubiquitin 98 32 17 −2.00 0.00 −1.57 0.00 −1.77 0.00 11 
              
Q07955 Splicing factor, arginine/serine-rich 1 98 30 15 −4.29 0.03 −5.98 0.22 0.03 0.37 
P11388 DNA topoisomerase II, α isozyme 92 24 10 −5.46 0.03 −6.36 0.18 0.19 0.18 
Q13283 Ras-GTPase-activating protein binding protein 1 98 27 15 −5.53  −4.70  0.25 0.43 
Q15019 Septin 2 (NEDD5 protein homologue) 10 98 31 18 −4.27 0.00 −3.79 0.00 0.77 0.00 
Q9NVA2 Septin 11 99 30 17 −4.50 0.01 −5.60 0.03 0.88 0.05 
Q16181 Septin 7 (CDC10 protein homologue) 14 98 31 15 −5.17 0.00 −5.70 0.00 1.01 0.00 
              
P61981 14-3-3 protein γ 99 30 15 −1.05 0.01 −1.08 0.60 −1.42 0.00 11 12 
P27986 PI3 kinase p85-α 99 26 15 −0.61  −1.82  −1.41  12 12 
Q16539 MAPK 14 (p38α) 10 98 29 14 −1.44 0.51 −1.52 0.57 −1.38 0.00 11 12 
P50990 T-complex protein 1, [thetas] subunit 98 28 15 −1.25 0.51 −0.63 0.88 −1.38 0.01 11 12 
P50897 Palmitoyl-protein thioesterase 1 precursor 98 25 13 −0.68 0.93 −1.22 0.28 −1.27 0.00 11 12 
P62258 14-3-3 protein epsilon 98 30 15 −1.22 0.00 −1.13 0.24 −1.25 0.00 11 12 
P63104 14-3-3 protein ζ/δ 98 30 15 −1.12 0.15 −1.34 0.05 −1.22 0.00 11 12 
              
O43353 Receptor-interacting serine/threonine protein kinase 2 (RIPK2) 99 27 17 −2.26  1.18  −4.31  24 
Q07912 Activated CDC42 kinase 1 (ACK-1) 99 38 19 −0.82 0.20 0.25 0.23 −2.78 0.02 14 24 
Q7Z4E9 MSTP089 98 31 16 −0.42 0.00 0.03 0.80 −2.36 0.00 15 24 
Q15370 Transcription elongation factor B polypeptide 96 27 11 −0.72 0.06 0.67 0.92 −2.08 0.00 16 24 
Q9UPX8 SH3 and multiple ankyrin repeat domains protein 2 (Shank2) 99 24 14 −0.54 0.01 0.72 0.56 −2.01 0.06 16 24 
              
Q13094 Lymphocyte cytosolic protein 2 (SLP-76) 98 26 14 0.13 0.27 −2.24 0.00 −3.34 0.00 15 16 
Q8TAR5 Programmed cell death 98 24 14 −0.38 0.04 −2.93 0.00 −2.83 0.00 16 
P51692 STAT 5B 99 33 19 −0.73  −3.45  −2.74  16 
Q9HC35 Echinoderm microtubule-associated protein-like 4 (EMAP-4) 99 31 16 −0.24 0.00 −1.94 0.00 −2.66 0.00 15 16 
Q9H939 Proline-serine-threonine phosphatase–nteracting protein 2 (PSTPIP2) 98 30 16 −0.55 0.37 −1.72 0.00 −2.58 0.00 15 16 
P29350 SHP-1 (protein tyrosine phosphatase, non–receptor type 6; PTP1C) 12 98 29 17 −0.13 0.47 −2.42 0.00 −1.93 0.00 15 16 
P56945 CRK-associated substrate (p130Cas) 12 97 28 16 −0.39 0.54 −0.73 0.33 −1.83 0.00 15 17 
P61026 Ras-related protein Rab-10 95 28 14 −0.48 0.05 −0.86 0.01 −1.75 0.07 15 17 
P07602 Proactivator polypeptide precursor 97 27 14 −0.61 0.02 −0.77 0.05 −1.72 0.00 15 17 
O75340 Programmed cell death protein 98 33 18 0.00 0.51 −0.72 0.00 −1.24 0.00 15 17 
P38606 Vacuolar ATP synthase catalytic subunit A, ubiquitous isoform 99 28 19 −0.11 0.66 −0.99 0.02 −0.96 0.23 15 17 
              
Q99952 Protein tyrosine phosphatase, non–receptor type 18 (BDP-1) 99 38 20 0.42  −2.01  −2.80  15 21 
P14317 LYN substrate 1 99 32 16 0.66 0.04 −1.54 0.00 −2.53 0.00 15 21 
Q14247 Src substrate cortactin (Amplaxin; oncogene EMS1) 99 31 18 0.40 0.39 −1.09 0.28 −2.46 0.01 15 21 
P16591 Tyrosine protein kinase FER 98 29 14 0.40 0.01 −1.22 0.00 −2.44 0.00 15 21 
P37802 Transgelin 2 (SM22-α homologue) 99 25 17 0.12 0.75 −1.61 0.07 −2.44 0.00 15 21 
Q06187 Tyrosine-protein kinase BTK 98 32 18 0.07 0.87 −1.48 0.02 −2.39 0.00 15 21 
P07332 Tyrosine-protein kinase Fes/Fps kinase 99 29 17 0.67 0.00 −1.97 0.00 −2.38 0.00 15 21 
Q9Y6W5 Wiskott-Aldrich syndrome protein family member 2 (WASP-2) 96 28 13 0.39 0.09 −0.94 0.15 −2.32 0.00 15 21 
Q9BX66 Ponsin (c-Cbl–associated protein) 99 60 40 0.04  −1.33  −1.86  15 21 
O95429 BAG family molecular chaperone regulator-4 99 25 14 −0.02 0.96 −1.62 0.07 −1.80 0.00 15 21 
P43405 Tyrosine protein kinase SYK 99 32 18 1.92 0.00 −1.00 0.09 −1.69 0.00 19 21 
              
NP_055423.1 Cytoplasmic FMR1 interacting protein 1 (KIAA0068) 10 98 33 17 0.29 0.38 −0.10 0.46 −2.31 0.00 15 23 
Q9NTK4 P53-inducible protein (hypothetical protein KIAA1168) 98 32 16 0.16 0.16 −0.14 0.09 −2.27 0.00 15 23 
AAH10132.1 GAP-associated tyrosine phosphoprotein p62 (Sam68) 98 30 14 −0.03 0.10 −0.26 0.49 −2.27 0.00 15 23 
P23458 Tyrosine protein kinase JAK1 99 26 14 −0.64 0.16 −0.40 0.84 −2.18 0.02 15 23 
O60613 15 kDa selenoprotein precursor 99 25 13 −0.08  −0.58  −2.04  15 23 
Q15438 Cytohesin 1 (SEC7 homologue B2-1) 98 30 16 −0.62 0.00 −0.35 0.93 −2.04 0.00 15 23 
Q9Y2A7 Nck-associated protein 1 (p125Nap1) 10 98 27 13 −0.20 0.40 −0.30 0.27 −1.89 0.00 15 23 
P49023 Paxillin 98 28 16 −0.37 0.23 −0.33 0.79 −1.83 0.00 15 23 
Q05397 Focal adhesion kinase 1 (FAK1) 10 98 35 19 −0.41 0.00 0.06 0.12 −1.76 0.00 16 23 
P26447 S100 calcium-binding protein A4 (metastasin) 97 29 12 −0.59 0.02 −0.44 0.11 −1.76 0.01 15 23 
Q14192 LIM domain protein DRAL 95 22 11 −0.32 0.56 0.09 0.83 −1.71 0.00 16 23 
AccProtein namePep%CScDst1 hP14 hP424 hP24ClSOM
P10721 Kit (mast/stem cell growth factor receptor precursor) 29 98 32 17 −3.57 0.00 −3.93 0.00 −3.81 0.00 11 
P27361 MAPK 3 (Erk1) 98 29 15 −2.99 0.03 −2.51 0.05 −3.35 0.02 11 
P28482 MAPK 1 (Erk2) 97 28 14 −2.71 0.00 −1.83 0.16 −2.92 0.00 11 
P62993 Grb2 (adapter protein) 99 33 19 −3.03 0.00 −0.95 0.00 −2.64 0.00 11 
P22681 CBL E3 ubiquitin protein ligase 98 28 14 −1.55 0.00 −1.46 0.01 −2.63 0.00 11 
P62988 Ubiquitin 98 32 17 −2.00 0.00 −1.57 0.00 −1.77 0.00 11 
              
Q07955 Splicing factor, arginine/serine-rich 1 98 30 15 −4.29 0.03 −5.98 0.22 0.03 0.37 
P11388 DNA topoisomerase II, α isozyme 92 24 10 −5.46 0.03 −6.36 0.18 0.19 0.18 
Q13283 Ras-GTPase-activating protein binding protein 1 98 27 15 −5.53  −4.70  0.25 0.43 
Q15019 Septin 2 (NEDD5 protein homologue) 10 98 31 18 −4.27 0.00 −3.79 0.00 0.77 0.00 
Q9NVA2 Septin 11 99 30 17 −4.50 0.01 −5.60 0.03 0.88 0.05 
Q16181 Septin 7 (CDC10 protein homologue) 14 98 31 15 −5.17 0.00 −5.70 0.00 1.01 0.00 
              
P61981 14-3-3 protein γ 99 30 15 −1.05 0.01 −1.08 0.60 −1.42 0.00 11 12 
P27986 PI3 kinase p85-α 99 26 15 −0.61  −1.82  −1.41  12 12 
Q16539 MAPK 14 (p38α) 10 98 29 14 −1.44 0.51 −1.52 0.57 −1.38 0.00 11 12 
P50990 T-complex protein 1, [thetas] subunit 98 28 15 −1.25 0.51 −0.63 0.88 −1.38 0.01 11 12 
P50897 Palmitoyl-protein thioesterase 1 precursor 98 25 13 −0.68 0.93 −1.22 0.28 −1.27 0.00 11 12 
P62258 14-3-3 protein epsilon 98 30 15 −1.22 0.00 −1.13 0.24 −1.25 0.00 11 12 
P63104 14-3-3 protein ζ/δ 98 30 15 −1.12 0.15 −1.34 0.05 −1.22 0.00 11 12 
              
O43353 Receptor-interacting serine/threonine protein kinase 2 (RIPK2) 99 27 17 −2.26  1.18  −4.31  24 
Q07912 Activated CDC42 kinase 1 (ACK-1) 99 38 19 −0.82 0.20 0.25 0.23 −2.78 0.02 14 24 
Q7Z4E9 MSTP089 98 31 16 −0.42 0.00 0.03 0.80 −2.36 0.00 15 24 
Q15370 Transcription elongation factor B polypeptide 96 27 11 −0.72 0.06 0.67 0.92 −2.08 0.00 16 24 
Q9UPX8 SH3 and multiple ankyrin repeat domains protein 2 (Shank2) 99 24 14 −0.54 0.01 0.72 0.56 −2.01 0.06 16 24 
              
Q13094 Lymphocyte cytosolic protein 2 (SLP-76) 98 26 14 0.13 0.27 −2.24 0.00 −3.34 0.00 15 16 
Q8TAR5 Programmed cell death 98 24 14 −0.38 0.04 −2.93 0.00 −2.83 0.00 16 
P51692 STAT 5B 99 33 19 −0.73  −3.45  −2.74  16 
Q9HC35 Echinoderm microtubule-associated protein-like 4 (EMAP-4) 99 31 16 −0.24 0.00 −1.94 0.00 −2.66 0.00 15 16 
Q9H939 Proline-serine-threonine phosphatase–nteracting protein 2 (PSTPIP2) 98 30 16 −0.55 0.37 −1.72 0.00 −2.58 0.00 15 16 
P29350 SHP-1 (protein tyrosine phosphatase, non–receptor type 6; PTP1C) 12 98 29 17 −0.13 0.47 −2.42 0.00 −1.93 0.00 15 16 
P56945 CRK-associated substrate (p130Cas) 12 97 28 16 −0.39 0.54 −0.73 0.33 −1.83 0.00 15 17 
P61026 Ras-related protein Rab-10 95 28 14 −0.48 0.05 −0.86 0.01 −1.75 0.07 15 17 
P07602 Proactivator polypeptide precursor 97 27 14 −0.61 0.02 −0.77 0.05 −1.72 0.00 15 17 
O75340 Programmed cell death protein 98 33 18 0.00 0.51 −0.72 0.00 −1.24 0.00 15 17 
P38606 Vacuolar ATP synthase catalytic subunit A, ubiquitous isoform 99 28 19 −0.11 0.66 −0.99 0.02 −0.96 0.23 15 17 
              
Q99952 Protein tyrosine phosphatase, non–receptor type 18 (BDP-1) 99 38 20 0.42  −2.01  −2.80  15 21 
P14317 LYN substrate 1 99 32 16 0.66 0.04 −1.54 0.00 −2.53 0.00 15 21 
Q14247 Src substrate cortactin (Amplaxin; oncogene EMS1) 99 31 18 0.40 0.39 −1.09 0.28 −2.46 0.01 15 21 
P16591 Tyrosine protein kinase FER 98 29 14 0.40 0.01 −1.22 0.00 −2.44 0.00 15 21 
P37802 Transgelin 2 (SM22-α homologue) 99 25 17 0.12 0.75 −1.61 0.07 −2.44 0.00 15 21 
Q06187 Tyrosine-protein kinase BTK 98 32 18 0.07 0.87 −1.48 0.02 −2.39 0.00 15 21 
P07332 Tyrosine-protein kinase Fes/Fps kinase 99 29 17 0.67 0.00 −1.97 0.00 −2.38 0.00 15 21 
Q9Y6W5 Wiskott-Aldrich syndrome protein family member 2 (WASP-2) 96 28 13 0.39 0.09 −0.94 0.15 −2.32 0.00 15 21 
Q9BX66 Ponsin (c-Cbl–associated protein) 99 60 40 0.04  −1.33  −1.86  15 21 
O95429 BAG family molecular chaperone regulator-4 99 25 14 −0.02 0.96 −1.62 0.07 −1.80 0.00 15 21 
P43405 Tyrosine protein kinase SYK 99 32 18 1.92 0.00 −1.00 0.09 −1.69 0.00 19 21 
              
NP_055423.1 Cytoplasmic FMR1 interacting protein 1 (KIAA0068) 10 98 33 17 0.29 0.38 −0.10 0.46 −2.31 0.00 15 23 
Q9NTK4 P53-inducible protein (hypothetical protein KIAA1168) 98 32 16 0.16 0.16 −0.14 0.09 −2.27 0.00 15 23 
AAH10132.1 GAP-associated tyrosine phosphoprotein p62 (Sam68) 98 30 14 −0.03 0.10 −0.26 0.49 −2.27 0.00 15 23 
P23458 Tyrosine protein kinase JAK1 99 26 14 −0.64 0.16 −0.40 0.84 −2.18 0.02 15 23 
O60613 15 kDa selenoprotein precursor 99 25 13 −0.08  −0.58  −2.04  15 23 
Q15438 Cytohesin 1 (SEC7 homologue B2-1) 98 30 16 −0.62 0.00 −0.35 0.93 −2.04 0.00 15 23 
Q9Y2A7 Nck-associated protein 1 (p125Nap1) 10 98 27 13 −0.20 0.40 −0.30 0.27 −1.89 0.00 15 23 
P49023 Paxillin 98 28 16 −0.37 0.23 −0.33 0.79 −1.83 0.00 15 23 
Q05397 Focal adhesion kinase 1 (FAK1) 10 98 35 19 −0.41 0.00 0.06 0.12 −1.76 0.00 16 23 
P26447 S100 calcium-binding protein A4 (metastasin) 97 29 12 −0.59 0.02 −0.44 0.11 −1.76 0.01 15 23 
Q14192 LIM domain protein DRAL 95 22 11 −0.32 0.56 0.09 0.83 −1.71 0.00 16 23 

NOTE: Proteins were quantitated by isobaric peptide labeling and LC-MS/MS. Proteins were clustered by log2 expression ratios (1, 4, and 24 hours) with respect to time using hierarchical clustering and self-organizing maps. Protein clusters identified by self-organizing maps and markedly regulated by inhibition of Kit kinase in a statistically significant (P < 0.05) manner are shown. A complete data set is in Supplementary Table S1 [available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/)].

Abbreviations: Acc, accession number; Pep, number of unique peptides; %C, mean peptide confidence; Sc, mean peptide score; Dst, mean peptide distance score to next best match; P, probability; Cl, hierarchical cluster; SOM, self-organizing map cluster.

Pharmacologic Changes in Kit Autophosphorylation and Substrate Phosphorylation

Cellular components involved in Kit signaling were measured by antiphosphotyrosine selection at multiple time points after inhibition of kinase activity by OSI-930. A rapid and reproducible loss of the pharmacologic target, the Kit receptor kinase, from the antiphosphotyrosine fraction was observed within the first hour of exposure to OSI-930, which was maintained throughout the 4- and 24-hour time points. The reduction in Kit interaction with the antiphosphotyrosine affinity resin, measured across 147 peptides (comprising 29 unique peptides), was significant (P < 0.0001) for the 1-, 4-, and 24-hour time points measured. The mean log2 expression ratios for Kit were −3.57 (±1.25), −3.93 (±1.43), and −3.81 (±1.26), respectively (Table 2), indicating a rapid and sustained attenuation of Kit kinase by OSI-930. Reductions in Kit autophosphorylation mediated by OSI-930 were also observed in separate biological experiments using fixed-time-point isobaric tags for relative and absolute quantitation (2 hours OSI-930: −2.05; P < 0.0001) and cleavable isotope-coded affinity tag (19) approaches (2 hours OSI-930: −3.03; P = 0.013). These data were also qualitatively in agreement with the rapid OSI-930–mediated loss of tyrosine phosphate observed by immunoblot with phosphospecific antibodies to Y721 or Y703 on Kit (Fig. 2A and B) and by antiphosphotyrosine immunoprecipitation followed by anti-Kit immunoblot (data not shown).

In addition to the effect of reduced Kit phosphotyrosine content on the abundance of Kit protein isolated by antiphosphotyrosine affinity selection, the phosphorylation state of Kit Y703 could be directly measured within the complex mixture of peptides (Fig. 3B). For example, the reduction in Kit phosphorylation following OSI-930 treatment was directly measured at the phosphopeptide QEDHAEAAL(phosphotyrosine)K (Y703 within the Kit precursor), a Grb2-binding site within the kinase insert domain. The ratio of phosphopeptide decreases between treatment and control samples were log2 −3.41 (±1.1), −5.01 (±1.2), and −5.40 (±1.0) at the 1-, 4-, and 24-hour time points, respectively (Fig. 3B). The loss of tyrosine phosphate within mutant Kit was rapid and pronounced with respect to the epidermal growth factor receptor where considerable phosphate remained on the receptor even after prolonged kinase inhibition (20). Kit tyrosine residue Y703, together with Y936, are the major binding sites for Grb2 (23), which can further recruit Cbl and Cbl-B to effect receptor degradation via the proteosomal pathway. Correspondingly, the reduction in Kit Y703 autophosphorylation was associated with a reduction in the abundance of Grb2 and Cbl proteins (Table 2) isolated by antiphosphotyrosine capture, reflecting a change in the SH2 domain–mediated interaction of Grb2 and Cbl with tyrosine phosphorylated Kit and/or a change in the Kit-mediated phosphorylation of Grb2 and Cbl following binding to Kit.

Constitutively active mutant Kit generates downstream signals via multiple pathways, which were inhibited by OSI-930. For example, the Ras-Raf-Mek-Erk mitogenic pathway was shown to be inhibited by a reduction in the phosphorylation of Erk1/2 (Table 2; Fig. 3B). Similarly, the Ras-GTPase–activating protein binding protein-1 was markedly down-regulated at the 1- and 4-hour time points (log2 −5.53 and −4.70, respectively; data not shown). The PI-3′ kinase survival pathway was shown to be perturbed by Kit kinase inhibition by a reduction in the PI-3′ kinase regulatory p85α subunit phosphorylation (log2 −0.61, −1.82, and −1.41) after 1, 4, and 24 hours of exposure to OSI-930, respectively (Table 2). Similarly, duplex isobaric tags for relative and absolute quantitation measurements comparing control and Kit inhibition after 2 hours exposure to OSI-930 showed a reduction in p85α of −1.39. This effect correlated with the observed reduction in phosphotyrosine content at the major binding site on Kit for the p85 subunit of PI-3′ kinase (Y721) by immunoblot analysis (Fig. 2A and B). The attenuation of PI-3 kinase pathway activity was further indicated by a decrease in serine-threonine phosphorylation of the downstream components Akt, S6K, and S6 in immunoblotting and immunohistochemical analyses (Fig. 2A and B; Table 1).

The transcription factors STAT-3 and STAT-5 can be phosphorylated through growth factor receptor activation, allowing α/β importin-dependent translocation to the nucleus and the transcription of genes required for cell cycle traverse. STAT5B showed a time-dependent decrease in abundance with Kit inhibition (log2 −0.73, −3.45, −2.74 at 1, 4, and 24 hours), and a decrease in tyrosine phosphorylation of STATs 3 and 6 was also observed by immunoblot and cell pellet microarray approaches (Fig. 2B; Table 1). The decrease in STAT phosphorylation would abrogate importin-dependent nuclear localization and STAT-dependent transcription of proproliferative and antiapoptotic genes (24). Whereas the temporal relationship between the reductions in abundance of Kit and STAT5B are consistent with phosphorylation of STAT5B being carried out directly by Kit, it is also possible that STAT5B was phosphorylated through Kit activation of Src and Fes/Fer family kinases. The temporal differences between JAK1 and STAT5B abundance suggest direct phosphorylation of STAT5B by JAK1 to be unlikely. These observations highlight an advantage of quantitative multiplex temporal analysis in allowing direct and indirect signaling relationships to be distinguished.

Expression Ratio Clustering of Proteins Regulated by Constitutive Kit Kinase Activity

Hierarchical clustering and self-organizing maps were used to identify additional phosphotyrosine and associated proteins whose interactions with the antiphosphotyrosine affinity resin were inhibited by OSI-930 with a similar time course to that observed for the pharmacologic target Kit. The use of clustering methods greatly simplified the data analysis of hundreds of proteins isolated by affinity selection, allowing a rapid focus on those protein sets with specific expression patterns and functions. Protein expression ratios, reflecting a measure of protein interaction with the antiphosphotyrosine affinity matrix, ranged from log2 −6.4 to 1.9 over the three time points. The clustering dendrogram was used to produce a temporal heat map of protein interactions with antiphosphotyrosine affinity resin (Fig. 4A), where the green color reflects a decrease of ≥log2 −2.5 and red color indicates an increase of ≥log2 2.0. Protein expression patterns were grouped using self-organizing maps and hierarchical clustering (Table 2). Those proteins most closely related to Kit in temporal phosphorylation pattern were the SH2 domain adapter Grb2, the MAPKs Erk-1 and Erk-2, and the E3 ubiquitin ligase c-Cbl and polyubiquitin (Fig. 4B). These proteins all showed a marked reduction in antiphosphotyrosine affinity within 1 hour of Kit inhibition, with profound and continued shutdown after 4 and 24 hours of exposure to inhibitor. Whereas gross perturbation of the Erk, PI-3 kinase, and STAT3/5B pathways might be expected following the blockade of a constitutively active receptor tyrosine kinase and in part serve to engender confidence in the methods used, additional regulators of Kit signaling were identified and measured. For example, the SH2 domain containing tyrosine phosphatase Shp-1 was shown to slightly increase after 1 hour followed by a rapid and marked decrease by 4 and 24 hours. The abundance of Shp-1 was consistent with its phosphorylation state. Shp-1 phosphorylation was directly measured on phosphopeptide EDV(phosphotyrosine)ENLHTK (Y566) where after 1 hour of exposure to OSI-930 an increase (log2) of 0.71 (±0.06) was observed, followed by a sharp decrease at 4 and 24 hours of −5.02 (±2.84) and −3.08 (±0.79), respectively (Fig. 3C). Self-organizing map group 16 included the tyrosine phosphatase Shp-1, SH2 adapter Slp-76, and “programmed cell death-4,” which showed slight up- or down-comodulation at 1 hour with marked down-regulation by 4 and 24 hours. Genetic studies with Kit null and tyrosine phosphatase Shp-1 null mice have implicated Shp-1 as a negative regulator of Kit function in vivo (25, 26); in vitro studies indicate that ubiquitin-mediated Shp-1 degradation may contribute to transformation by Kit mutation (27). The phosphorylation of Shp-1 has been shown to be important for maximal dephosphorylation of substrates, and consistent with this model mutation of Shp-1 Y538 and Y566 were shown to impair its function (28). The PEST domain tyrosine phosphatase BDP-1 (29, 30) shared a similar temporal phosphorylation profile following Kit inhibition. A slight increase in BDP-1 of log2 0.42 after 1 hour Kit inhibition, followed by a sharp decrease of −2.01 and −2.80 after 4 and 24 hours, respectively, was observed. BDP1 has been shown to negatively regulate erbB2 phosphorylation, correlating with the dephosphorylation of the Grb2-associated protein Gab1 and a reduction in the activity of Erk2 (30). The interaction between Shp-1 and/or BDP-1 and Kit would account for the rapid dephosphorylation of Kit following kinase inhibition. The protein tyrosine phosphatase BDP1; the nonreceptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk; the Lyn kinase substrate HS1; the Src substrate cortactin; the Cbl-associated protein ponsin; and the cytoskeletal adapter protein WASP were coclustered in self-organizing map-21. These proteins showed slight up- or down-modulation at 1 hour with less down-regulation by 4 hours than the Kit cluster self-organizing map-11. The non–receptor tyrosine kinase Syk was markedly up-regulated 1 hour after addition of OSI-930 (log2 1.92), potentially representing a homeostatic response to the removal of the major Kit tyrosine kinase signal from the cell. Interestingly the dual Bcr-Abl/Kit inhibitor STI571 also was shown to transiently stimulate tyrosine phosphorylation of Syk in the myeloid leukemia line K562 (31), indicating the up-regulation of Syk upon Kit inhibition was more general and not specific to the mast cell leukemia line HMC-1.

Figure 4.

A, heat map of hierarchically clustered proteins allowed grouping of proteins showing similar temporal expression patterns and indicated inhibition of Kit kinase activity down-regulated antiphosphotyrosine selection of substrates and substrate-interacting proteins in a time-dependent manner. The green color reflects a decrease of ≥log2 −2.5 and red color indicates an increase of ≥log2 2.0. Time of exposure to OSI-930 (500 nmol/L) is reflected in the X axis. Specific hierarchical clusters and self-organizing map clusters were referenced in Table 2 and Supplementary Table S1 [available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/)]. B, self-organizing mapping of expression ratios indicating clustering of key Kit target proteins coregulated through OSI-930–mediated kinase inhibition. Expression ratios (log2; control versus treatment) are indicated on the Y axis. Time of exposure to OSI-930 (500 nmol/L) is indicated on the X axis.

Figure 4.

A, heat map of hierarchically clustered proteins allowed grouping of proteins showing similar temporal expression patterns and indicated inhibition of Kit kinase activity down-regulated antiphosphotyrosine selection of substrates and substrate-interacting proteins in a time-dependent manner. The green color reflects a decrease of ≥log2 −2.5 and red color indicates an increase of ≥log2 2.0. Time of exposure to OSI-930 (500 nmol/L) is reflected in the X axis. Specific hierarchical clusters and self-organizing map clusters were referenced in Table 2 and Supplementary Table S1 [available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/)]. B, self-organizing mapping of expression ratios indicating clustering of key Kit target proteins coregulated through OSI-930–mediated kinase inhibition. Expression ratios (log2; control versus treatment) are indicated on the Y axis. Time of exposure to OSI-930 (500 nmol/L) is indicated on the X axis.

Close modal

Receptor tyrosine kinases have been shown to regulate the assembly and disassembly of cellular contacts required for cell migration and division (32, 33). Components of actin filament adhesion complexes (34), e.g., paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, WASP, cdc42, FHL-3, ACK-1, actin, cortactin, NAP1, CAP-G, zyxin, and SH3P12/ponsin were identified within the phosphotyrosine fraction. These proteins showed modest decreases in antiphosphotyrosine selection associated with Kit inhibition except at the 24-hour time point when HMC-1 cell apoptosis became evident and significant reductions in recovery were apparent. For example, expression ratios within the phosphotyrosine fraction at the 1-, 4-, and 24-hour time points were as follows: paxillin (log2 −0.37, −0.33, and −1.83), p130CAS (−0.39, −0.73, and −1.83), and FAK (−0.41, −0.06, and −1.76); these expression changes achieved significance by the 24-hour time points (P < 0.01). The reduction in phospho-paxillinY118 through Kit inhibition was also observed by cell pellet tissue microarray immunohistochemistry (Table 1). These data suggest that inhibition of Kit activity by OSI-930 exerted a negative effect on the assembly of focal adhesion complexes over time.

Here, we have used a small-molecule inhibitor of the Kit receptor tyrosine kinase, OSI-930, together with temporal expression clustering to allow rapid definition of those proteins physiologically regulated by Kit kinase activity. Attenuation of Ras, PI-3′ kinase, and STAT signaling pathways were measured by affinity LC-MS/MS, by immunoblot, and by tissue microarrays of fixed cell pellets, with comparable results and served to validate the affinity LC-MS/MS protein identification and quantitation approach. Modulation of the phosphorylation of Kit on Y703 and the tyrosine phosphatase Shp-1 Y566 was directly measured within complex peptide mixtures. The Kit kinase was shown to modulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2, which may explain the rapid dephosphorylation of Kit upon kinase inhibition in contrast to other receptor tyrosine kinases, such as the epidermal growth factor receptor, where the dephosphorylation rate can be much slower (20). Functional crosstalk between non–receptor tyrosine kinases and Kit following inhibition of Kit kinase activity was measured as a function of time, where Syk phosphorylation was markedly up-regulated. Phosphorylation-dependent Kit crosstalk with focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, WASP, FHL-3, cdc42, ACK-1, cortactin, NAP1, SH3P12/ponsin) and septin-actin (NEDD5, cdc11, actin) assemblies was observed and supports the integration of cell proliferation and survival signals with those regulating cell adhesion and migration. The combined use of isobaric labeling, immunoblot, and tissue microarray strategies allowed the rapid and sensitive identification of proteins involved in Kit signaling as well as their temporal measurement in the HMC-1 cell line model of mast cell leukemia. The approach described is potentially applicable to analysis of temporally defined biological cellular process in vitro or in vivo and to the identification of biomarkers associated with physiologic responses to small molecule inhibitors.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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