Synthesis, Chaperoning, and Metabolism of Proteins Are Regulated

Synthesis, Chaperoning, and Metabolism of Proteins Are Regulated by NT-3/TrkC Signaling in the Medulloblastoma Cell Line DAOY Mariella Gruber-Olipitz,‡ Thomas Ströbel,§ Wei-Qiang Chen,‡ Michael A. Grotzer,| Franz Quehenberger,⊥ Irene Slavc,*,‡ and Gert Lubec‡,† Department of Pediatrics, Medical University of Vienna, Vienna, Austria, Institute of Neurology, Medical University of Vienna, Vienna, Austria, Department of Pediatrics, University Children’s Hospital Zurich, Zurich, Switzerland, and Institute of Medical Informatics, Statistics and Documentation, Medical University of Graz, Graz, Austria Received November 09, 2007

The human medulloblastoma cell line DAOY was transfected with Tropomyosin receptor kinase (TrkC), a marker for good prognostic outcome. Following TrkC-activation by its ligand neurotrophin-3, protein extracts from DAOY cells were run on 2DE with subsequent MALDI-TOF-TOF analysis and quantification in order to detect downstream effectors. Protein levels of translational, splicing, processing, chaperone, protein handling, and metabolism machineries were shown to depend on neurotrophin-3-induced TrkC activation probably representing pharmacological targets. Keywords: TrkC • neurotrophin-3 • medulloblastoma • 2D gel electrophoresis • MALDI-TOF-TOF • protein machinery

Introduction Medulloblastoma (MB) is the most frequent malignant brain tumor in childhood. Recent reports of two independent groups identified high neurotrophin receptor TrkC mRNA expression as a powerful independent predictor of favorable survival outcome in MB patients.1 It remains to be determined, however, which downstream mechanisms and molecules mediate such favorable tumor characteristics. Neurotrophins are a family of related trophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3, NT 4/5, and NT 6. They regulate proliferation, differentiation, and death of neuronal progenitor cells by binding one or more members of a family of high affinity neurotrophin receptor tyrosine kinases, that is, TrkA, TrkB, or TrkC (previously described as an oncogene called tropomyosin receptor kinase). Neurotrophin-3 specifically activates TrkC and has been shown to promote differentiation, survival, and proliferation of sensory and motor neurons, cerebellar granule cells, and peripheral nervous system precursors.2 Several distinct signal transduction pathways are known to mediate these biological effects. Binding of neurotrophins to Trk receptors causes receptor dimerization, activation of the intrinsic tyrosine kinase, and receptor trans-autophosphorylation. Phosphorylation of cytoplasmic tyrosines creates rec* To whom correspondence should be addressed. Prof. Irene Slavc, Medical University of Vienna, Dpt. of Pediatrics, Waehringer Guertel 18-20, 1090 Vienna, Austria. Tel: x 43-1-40400-3232. Fax: x43-1-40400-3093. E-mail: [email protected]. § Institute of Neurology, Medical University of Vienna. | University Children’s Hospital Zurich. ⊥ Medical University of Graz. ‡ Department of Pediatrics, Medical University of Vienna. † Work was carried out in the laboratory of G.L.

1932 Journal of Proteome Research 2008, 7, 1932–1944 Published on Web 03/13/2008

ognition sites for adaptor proteins that link these receptors to intracellular signaling cascades. Among them, the Ras-mitogen activated protein kinase (MAPK) pathway, the phosphatidylinositol-3-OH kinase (PI3K)/Akt kinase pathway, and phospholipase C-γ (PLC- γ1) are activated. While a series of biological effects and activated signaling pathways are known to be activated by NT-3/TrkC, the effects on regulation of RNA processing, splicing, and translation, as well as protein chaperoning, handling, and metabolism, in medulloblastoma remain elusive. Studying these functions is of pivotal interest since these individual steps are essential for tumor biology: disruption in one or more of the steps that control protein biosynthesis has been associated with alteration and the regulation of cell growth and cell cycle progression.3,4 Tight regulation of the multiple steps from mRNA to protein networks is mandatory to the process of malignant transformation: changes in mRNA splicing have been shown to play a functionally significant role in tumor biology,5 and there is increasing evidence that deregulation of gene expression at the level of mRNA translation can contribute to cell transformation and the development of a malignant phenotype.6,7 Once protein synthesis is completed, chaperones guide normal folding, intracellular disposition, and proteolytic turnover of many key regulatory proteins in cell growth, differentiation, and survival. This essential guardian function was also described as being subverted during oncogenesis allowing malignant transformation.8 Finally, as controlled intracellular protein degradation is crucial for maintenance of normal cell function and is also involved in growth control, deregulations in this process may also contribute to carcinogenesis.9,10 Here we aimed to search for TrkC-dependent proteins reflecting individual functions of the protein machinery using 10.1021/pr700724a CCC: $40.75

 2008 American Chemical Society

Proteins’ Synthesis/Chaperoning/Metabolism Regulated by NT-3/TrkC a proteomic approach. The observed aberrant levels of individual proteins from different functional protein classes indicate derangement of protein networks linked to specific TrkC receptor activation.

Experimental Section Cell Culture. DAOY/empty vector clone 2A4 and DAOY/TRK clone 1G4 (kindly provided by Dr. M. Grotzer) were maintained at 37 °C in a humidified atmosphere with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 4 mM L-glutamine, penicillin (100 units/mL), streptomycin (100 µg/ mL), and G418 (250 µg/mL). For the NT-3 induction experiment, DAOY transfectants were washed twice with Hanks’ buffered salt solution followed by adaptation to serum-free medium (DMEM supplemented with 1% nonessential amino acids, 4 mM L-glutamine, penicillin (100 units/mL), streptomycin (100 µg/mL), 1 µg/mL transferrin, 30 nM sodium selenite, 20 nM progesterone, 100 µM putrescine, and 1 µg/mL insulin) for 24 h. The following day, the cells were incubated with 100 ng/mL NT-3 (R&D Systems, Minneapolis, MN) known to activate TrkC (unpublished results) and harvested at time points 0, 6, 12, 24, and 48 h (Supplemental Figure 1 in Supporting Information). These time points were chosen to cover immediate as well as late responses to TrkC activation. To allow for statistically robust analyses, five independent experiments were carried out. Cells were scraped from dishes, washed three times in 2 mL of ice-cold phosphate buffered saline (Gibco BRL, Gaithersburg, MD), and centrifuged 3 min at 4 °C and 1000g. The cell pellet was flash-frozen in liquid nitrogen and stored at -80 °C, and the freezing chain was never interrupted. RT-PCR studies revealed a 32 000-fold increase of TrkC expression levels in transfected cells as compared to empty vector cells (data not shown), and this level was kept during the testing period. Sample Preparation. The cell pellet was suspended in 1.0 mL of sample buffer consisting of 7 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Sigma, St. Louis, MO), 4% CHAPS (3[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma, St. Louis, MO), 65 mM 1,4-dithioerythritol (Merck, Germany), 1 mM EDTA (ethylenediaminetraacetic acid) (Merck, Germany), protease inhibitors complete (Roche, Basel, Switzerland), and 1 mM phenylmethylsulfonyl chloride. The suspension was sonicated for approximately 15 s on ice. After homogenization, samples were left at room temperature for 1 h and centrifuged at 14 000 rpm for 1 h. The supernatant was transferred into Ultrafree-4 centrifugal filter units (Millipore, Bedford, MA) for desalting and concentrating proteins. Protein content of supernatants was quantified by Bradford protein assay system.11 The standard curve was generated using bovine serum albumin, and absorbance was measured at 595 nm. Two-Dimensional Gel Electrophoresis (2-DE). Samples prepared from each cell line were subjected to 2-DE as described elsewhere.12 One milligram of protein was applied on immobilized pH 3–10 nonlinear gradient strips at their basic and acidic ends. Focusing was started at 200 V, and voltage was gradually increased to 8000 V over 31 h and then kept constant for a further 3 h (approximately 150 000 Vh totally). After the first dimension, strips (18 cm) were equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS, and 2% DTT

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and subsequently for 15 min in the same buffer containing 2.5% iodo-acetamide instead of DDT. After equilibration, strips were loaded on 9–16% gradient sodium dodecylsulfate polyacrylamide gels for second-dimensional separation. Gels (180 × 200 × 1.5 mm) were run at 40 mA per gel. Immediately after the second dimension run, gels were fixed for 18 h in 50% methanol, containing 10% acetic acid; gels were then stained with Colloidal Coomassie Blue (Novex, San Diego, CA) for 12 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories, Hercules, CA) covering the range 10–250 kDa. pI values 3–10 were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess of dye was washed out from gels with distilled water, and gels were scanned with ImageScanner (Amersham Bioscience). Electronic images of the gels were recorded using Adobe Photoshop and Microsoft Power Point Softwares. MALDI-TOF and MALDI-TOF/TOF Mass Spectrometry (MS). Spots were excised with a spot picker (PROTEINEER sp, Bruker Daltonics, Bremen, Germany) and placed into 96-well microtiter plates, and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dp, Bruker Daltonics).12 Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/µL trypsin (Promega, Madison, WI) in enzyme buffer (consisting of 5 mM octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 h at 30 °C. Peptide extraction was performed with 10 µL of 1% TFA in 5 mM OGP. Extracted peptides were directly applied onto a target (AnchorChip, Bruker Daltonics) that was loaded with R-cyano-4hydroxy-cinnamic acid (Bruker Daltonics) matrix thinlayer. The mass spectrometer used in this work was an Ultraflex TOF/ TOF (Bruker Daltonics) operated in the reflector mode for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF with a fully automated mode using the FlexControl software. An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M + H]+ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1–17 and clip 18–39). Each spectrum was produced by accumulating data from 200 consecutive laser shots for PMF. Those samples which were analyzed by PMF from MALDI-TOF were additionally analyzed using LIFT-TOF/TOF MS/MS from the same target using two MS/MS modes: laser-induced dissociation (LID) and collision-induced dissociation (CID). In the LID-MS/MS mode using a long-lifetime N2 laser, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation in the TOF1 stage. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analyzed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd., London, U.K.). Database searches, through Mascot, using combined PMF and MS/MS data sets were performed via BioTools 2.2 software. A mass tolerance of 25 ppm and MS/ MS tolerance of 0.5 Da and 0 missing cleavage site were allowed, and oxidation of methionine residues was considered. Journal of Proteome Research • Vol. 7, No. 5, 2008 1933

research articles The probability score calculated by the software was used as criterion for correct identification (http://www.matrixscience. com/help/scoring_help.html). Protein Identification by NanoLC-ESI-MS/MS. A total of 6.4 µL of extraction solution was used for nanoLC-MS/MS investigation. The HPLC used was an Ultimate 3000 system (Dionex Corporation; Sunnyvale, CA) equipped with a PepMap C-18 analytic column (75 µm × 150 mm). The gradient was (A ) 0.1% formic acid in water, B ) 80% ACN/0.08% formic acid in water) 4% B to 60% B from 0 to 30 min, 90% B from 30 to 35 min, 4% B from 35 to 60 min. Peptide spectra were recorded over the mass range of m/z 350–1600, and MS/MS spectra were recorded in information-dependent data acquisition over the mass range of m/z 50–1600. Repeatedly, one MS spectrum was recorded followed by two MS/MS spectra on the QSTAR XL instrument; the accumulation time was 1 s for MS spectra and 2 s for MS/MS spectra. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. Doubly or triply charged peptides were chosen for MS/MS experiments due to their good fragmentation characteristics. MS/MS spectra were interpreted by the MASCOT software (Matrix Science, Boston, MA) and searched against UniProtKB 51.5 database to identify protein spot. The searching parameters were set as follows: a mass tolerance of 500 ppm for MS tolerance, 0.2 Da for MS/MS tolerance, one missing cleavage site, fixed modification of carbamidomethyl (C), and variable modification of methionine oxidation, phosphorylation(STY). A second error tolerant search was done using no enzyme to detect unspecific cleavage and unpredicted modifications. All identified modifications were manually rechecked. Quantification. Protein spots were outlined (first automatically and then manually) and quantified using the Proteomweaver software (Bio-Rad, Germany). The percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2-DE gel.13 Statistical Analysis. Values used for statistical analyses were expressed as means ( standard deviation (SD) of percentage of the spot volume in each particular gel after subtraction of the background values. Analyses applied included the global test by Goeman, logarithms of protein intensities at each time point were tested for group differences with Student’s t test, and a false discovery rate (FDR) of less than 0.05 was used to indentify differentially expressed proteins.14

Results and Discussion To identify specific protein expression changes following NT3-mediated TrkC activation in medulloblastoma cells, a partial proteome of a TrkC-transfected human medulloblastoma cell line DAOY and empty vector control cells was analyzed. To detect early and late effects of TrkC activation, five different time points between 0 and 48 h following ligand addition were selected. Five independent experiments were performed allowing statistical analyses running seven gels per time point and group, that is, a total of 70 2-D gels. A representative gel is shown in Figure 1. Identification of proteins from the protein machinery was carried out by MALDI-TOF or MALDI-TOF-TOF, and the majority of proteins with differential protein levels (nine out of thirteen) was validated by nano-ESI-LC-MS/MS (Q-TOF; 1934

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Gruber-Olipitz et al. Table 1). Because of technical reasons and/or limitation of protein available, four proteins could not be identified by Q-TOF. Quantification of spot volumes revealed 13 individual proteins from protein synthesis, chaperoning, and metabolism with different levels following TrkC activation (Figures 1 and 2, Table 2). The levels of 10 proteins were increased and the levels of three proteins were decreased by TrkC activation. As shown in the Table 2, early, late, and sustained protein expression was considered. Most TrkC-dependent proteins (n ) 9) showed differential levels at late time points, that is, at 24-48 h following TrkC activation, four proteins presented with lower levels at early time points (12 h or less), and two proteins showed differential expression over the whole time course (Table 2). NT-3/TrkC activation altered levels of seven proteins from the protein synthetic machinery: heterogeneous nuclear ribonucleoprotein H and K, probable rRNA processing protein EBP2, eukaryotic translation initiation factor 4H and 5A, U2 small nuclear RNA auxiliary factor 2, and 40S ribosomal protein SA. NT-3/TrkC controlled protein levels of the chaperones heat shock protein beta-1, T-complex protein 1, gamma subunit, and heat shock 70 kDa protein 1. Two components of the protein degradation machinery, 26S proteasome non-ATPase regulatory subunit 13 and cathepsin D, were NT-3/TrkC-dependent. Levels of the multifunctional 40S ribosomal protein (syn: 34/ 67 kDa laminin receptor; Colon carcinoma laminin-binding protein; Multidrug resistance-associated protein MGr1-Ag; 40S, p40, NEM/1CHD4) were elevated from 6 to 48 h in the present study following NT-3/TrkC activation as compared to empty vector controls. 40S was significantly elevated in all of the three individual spots probably representing splice variants or posttranslational modifications (Table 2). 40S is a ribosomal protein that acquired the novel function of laminin receptor activity during evolution.15 It induces down-reguation of MKP-1 expression, an event mandatory for oncogenesis, has been consistently observed in invasive and metastatic cancer cells, promotes tumor progression, and is associated with poor prognosis.16 This is not in disagreement with the observation that TrkC is linked to a good prognostic outcome (in vivo)1 because we cannot draw any conclusion on dignity from our in vitro studies. 40S downregulation was shown to significantly enhance cytotoxicity of anticancer drugs in gastric cancer cells,17 and a correlation between its expression and the degree of differentiation, invasiveness, and metastatic abilities in colorectal cancer cells could be established.18 Morphological assessment of differentiation, proliferation, and apoptosis up to 48 h (unpublished results), at time points when 40S levels were increased by approximately a factor of 4 in the present study, showed no changes of these parameters. Different time points, as well as different cell types, may account for the discrepancy. Although 40S acts as regulator of signaling systems as, for example, decreasing the extent of ERK, JNK, and p38 phosphorylation, the biological meaning of the 4-fold increase of 40S protein levels in NT-3/TrkC-activated DAOY cells over the observation period remains open, and it may be simply a bystander phenomenon, because above-mentioned tumor biological criteria including differentiation, proliferation, and apoptosis were comparable between controls and NT-3/TrkC cells. Studies are ongoing in our laboratory to study functional consequences of 40S overexpression in several medulloblas-

U2 (RNU2) small nuclear RNA auxiliary factor 2. isoform b

26S proteasome non-ATPase regulatory subunit 13 Heterogeneous nuclear ribonucleoprotein K

Cathepsin D

Heterogeneous nuclear ribonucleoprotein H

40S ribosomal protein SA

protein name

18

21

193

98

15

18

16 16 10 10

180

88

101 147 72 70

44

45

70

54

50 55 44 28

1

1

1

1

1 1 1 1

MS matching sequ. MS/MS score peptides cov. (%) peptides

MALDI TOF-TOF

53121

50976

42918

44552

49484

32854

theor. MW

9.2

5.4

5.5

6.1

5.9

4.8

MS/MS peptides

6.0

5.4

5.7

5.6

377

610

675

26

28

39

196NFAFLEFR203 204SVDETTQAMAFDGIIFQGQSLK225 277ELLTSFGPLK286 414SIEIPRPVDGVEVPGCGK431 432IFVEFTSVFDCQK444

87ILSISADIETIGEILK102 87ILSISADIETIGEILKK103 149LLIHQSLAGGIIGVK163 208IILDLISESPIK219 306NLPLPPPPPPR316 378GSYGDLGGPIITTQVTIPK396 397DLAGSIIGK405 423IDEPLEGSEDR433 434IITITGTQDQIQNAQYLLQNSVK456 175LGGLTQAPGNPVLAVQINQDK195

70TDYNASVSVPDSSGPER86

263DLNYCFSGMSDHR275 276YGDGGSTFQSTTGHCVHMR294 300ATENDIYNFFSPLNPVR316 327VTGEADVEFATHEDAVAAMSK347 356YVELFLNSTAGASGGAYEHR375 185QPGITFIAAK194 195FDGILGMAYPR205 206ISVNNVLPVFDNLMQQK222 223LVDQNIFSFYLSR235 268AYWQVHLDQVEVASGLTLCK287 288EGCEAIVDTGTSLMVGPVDEVR309 314AIGAVPLIQGEYMIPCEK331 332VSTLPAITLK341 349LSPEDYTLK357 393YYTVFDR399

PTMs

Oxidation (M212)

Deamidation (Q220)

Methylation (S216)

Deamidation (Q185) Oxidation (M201, M219, M301, M326)

Methylation (D65, E92, E163) Oxidation (M93, M271, M345)

Up-Regulated in DAOY-TrkC 4.7 4.8 4.6 5.4 481 28 234GAYGGGYGGYDDYNGYNDGYGFGSDR259 Acetylation(M2)

theor. observed MS/MS sequ. pI pI score cov (%)

nanoLC-ESI-MS/MS

Table 1. Identification and Characterization of NT-3/TrkC-Dependent Synthesis, Chaperoning, and Metabolism of Proteins in DAOY Medulloblastoma Cell Linea

Proteins’ Synthesis/Chaperoning/Metabolism Regulated by NT-3/TrkC

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Journal of Proteome Research • Vol. 7, No. 5, 2008 1935

1936

225

83

97

104

Heat-shock protein beta-1

Eukaryotic translation initiation factor 4H

Eukaryotic translation initiation factor 5A

68

MS score

Heat shock 70 kDa protein 1

Probable rRNA processing protein EBP2

protein name

Table 1. Continued


14

11

12

32

8

matching peptides

62

57

43

53

23

sequ. cov. (%)

MALDI TOF-TOF

1

1

1

1

1

MS/MS peptides

16701

27385

22783

70052

34820

theor. MW

6.1

observed pI

MS/MS score

sequ. cov (%) MS/MS peptides

5.1

6.7

6.0

5.1

6.3

6.0

724

201

475

66

14

52

56VHLVGIDIFTGK67 56VHLVGIDIFTGKK68 68KYEDICPSTHNMDVPNIK85 69YEDICPSTHNMDVPNIK85 86RNDFQLIGIQDGYLSLLQDSGEVR109 87NDFQLIGIQDGYLSLLQDSGEVR109 110EDLRLPEGDLGK121

97EALTYDGALLGDR109 152DDFNSGFR159 28NGFVVLK34

13GPSWDPFR20 13GPSWDPFRDWYPHSR27 28LFDQAFGLPR37 80QLSSGVSEIR89 97VSLDVNHFAPDELTVK112 141KYTLPPGVDPTQVSSSLSPEGTLTVEAPMPK171 83GFCYVEFDEVDSLK96

113AFYPEEISSMVLTK126 172IINEPTAAAIAYGLDR187 221ATAGDTHLGGEDFDNR236 237LVNHFVEEFKR247 300ARFEELCSDLFR311 302FEELCSDLFR311 326LDKAQIHDLVLVGGSTR342 329AQIHDLVLVGGSTR342 349LLQDFFNGR357 362SINPDEAVAYGAAVQAAILMGDK384 424QTQIFTTYSDNQPGVLIQVYEGER447 540NALESYAFNMK550 5RVPFSLLR12

Down-Regulated in DAOY-TrkC 5.5 5.5 795 31 4AAAIGIDLGTTYSCVGVFQHGK25

10.1

theor. pI

nanoLC-ESI-MS/MS

Oxidation (M79, M141)

Acetylation (A2)

Deamidation (Q224)

Acetylation (A2)

Oxidation (M169)

Deamidation (Q152, Q175)


Deamidation (Q330, Q376)

PTMs

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129ALDDMISTLK138 168WSSLACNIALDAVK181 204IPGGIIEDSCVLR216 238IVLLDSSLEYK248 295GISDLAQHYLMR306 331IVSRPEELREDDVGTGAGLLEIK353 400NVLLDPQLVPGGGASEMAVAHALTEK425 428AMTGVEQWPYR438 439AVAQALEVIPR449 492ELGIWEPLAVK502 508TAVETAVLLLR518 MW ) molecular weight; sequ. cov. ) sequence coverage; PTMs ) post-translational modifications. a

PTMs MS/MS peptides

49MLLDPMGGIVMTNDGNAILR68 43 847 6.0 6.1 60534 1 49 19 112 T-complex protein 1 gamma subunit

protein name

MS score

matching peptides

sequ. cov. (%)

MS/MS peptides

theor. MW

theor. pI

observed pI

MS/MS score

sequ. cov (%)

nanoLC-ESI-MS/MS MALDI TOF-TOF

Table 1. Continued



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toma cell lines. Unfortunately, no genetically manipulated mice or data from 40S in vitro overexpression in nonmalignant cell lines are available to predict a functional role in medulloblastoma cells. Moreover, assignment of functional roles for this protein are hampered by contradictions in nomenclature: a caution is given in the UniProtKB entry P08865 on July 10, 2007 (http://www.expasy.org/uniprot/P08865) indicating that 40S is no longer considered a laminin receptor. Protein levels of heterogeneous nuclear ribonucleoprotein H (hnRNPH) were increased following TrkC activation. hnRNPH was acetylated at M2 and methylated at D65, E92, and E163, and levels were increased about 3-fold from 6 to 48 h. While methylation may be a post-translational modification with probable biological consequences or simply a technical artifact, the meaning of M2 acetylation remains to be elucidated; M2 acetylation has been, however, already described for hnRNPH by mass spectrometry in another malignancy, B-cell lymphoma (P31943; http://www.expasy.org/uniprot/P31943). hnRNPH is implicated in several steps of pre-mRNA processing and in cellular differentiation.19 hnRNPH overexpression was observed in primary carcinomas and metastases.20 In vitro adminstration as well as overexpression of hnRNPH leads to production of proapoptotic Bcl-x(S), an alternative splice variant of Bcl-x in HeLa cells.21 At the studied time points, no increased apoptosis was observed following TrkC activation resulting in increased hnRNPH, again pointing to the specificity of signaling cascades. It may well be, however, that apoptosis is occurring at a later time point and increased hnRNPH levels are preceding this process which would be in line with evidence for a proapoptotic role of TrkC activation in DAOY cells.22 hnRNPH is interacting with N-myc-downstream-regulated gene 1 protein,23 and indeed, N-myc is a key element for medulloblastoma oncogenesis.24 Heterogeneous nuclear ribonucleoprotein K (hnRNPK) levels were increased at 48 h exclusively in the NT-3/TrkC activated DAOY cells, and development of levels at later time points cannot be predicted. This is meaningful for design of future studies on protein expression following TrkC activation considering inclusion of later time points. hnRNPK is an ancient RNA/DNA-binding protein involved in many processes of gene expression acting as a transcriptionalinducer and translational-regulator.25 By directly regulating mRNA export, splicing, mRNA stability, and initiation of translation, hnRNPK serves as a regulator of multiple steps in cell cycle progression and growth regulation.26,27 It is activated by the MAPK (ERK1/2) pathway, directly increasing MYC mRNA translation, thereby enhancing proliferation of leukemic blasts.28 Overexpression of hnRNPK in breast cancer cells has already been related to epidermal growth factor signaling and c-myc promoter activity and expression, leading to enhanced cell proliferation.29 In agreement with these observations, in medulloblastoma cells, c-myc downregulation induces growth suppression, cell cycle arrest, and apoptosis.30 Both, TrkC and c-myc are wellestablished prognostic factors in medulloblastoma, and herein, we provide evidence for a possible link between these reciprocal predictors. Translation initiation factor eIF4H (eIF4H) was increased doublefold following TrkC activation (represented by 1 spot) and showed A2 acetylation in agreement with previous work.31,32 The biological meaning of acetylation/deacetylation for activation or inactivation of proteins in tumor cells remains open, although acetylation per se as, for example, by deacetylase Journal of Proteome Research • Vol. 7, No. 5, 2008 1937

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Gruber-Olipitz et al.

Figure 1. 2-DE gel image of the human medulloblastoma cell line DAOY-TrkC. Proteins were extracted and separated on an immobilized pH 3–10 nonlinear gradient strip followed by separation on a 9–16% gradient polyacrylamide gel. Gels were stained with Coomassie blue and spots analysed by MALDI TOF-TOF and nano-LC-ESI-MS/MS.

inhibitors is a focus of tumor biological research. eIF4H stimulates helicase activity of eIF4A and functions in translation initiation through protein–protein interactions that stabilize conformational changes of eIF4A occurring during RNA binding and RNA duplex unwinding.33,34 Increased eIF4H levels may be relevant for impairing the stoichiometry of translationally active complexes that in turn may be responsible for aberrant translation processes observed in tumors.6 Probable rRNA processing protein EBP2 (syn: EBNA1-binding protein 2; Nucleolar protein p40; EBP2) is a nucleolar protein required for processing 27S pre-rRNA and thus essential for ribosome biogenesis and growth.35,36 EBP2 has been described to play a role in chromosome segregation in yeast37 and is differentially expressed in carcinomas at the nucleic acid level.38 No specific function has been assigned to EBP2 in tumor biology, and no regulation of EBP2 levels has been reported so far. Increased levels at late time points indicate that EBP2 is TrkC-dependent. U2 small nuclear RNA auxiliary factor 2 is a structure that was published at the nucleic acid (cDNA) level only.39 No functional roles were assigned to this protein, and molecular functions as nucleotide binding, RNA binding, and mRNA processing are simply inferred from electronic annotation from InterPro (http://www.ebi.ac.uk/interpro/). Herein evidence for its existence at the protein level in human cells (Table 1) is provided, and increased levels from 12 h following NT-3/TrkC activation were observed (Table 2). High identity (82%) to splicing factor U2AF 65 kDa subunit (Supplemental Figure 2 in Supporting Information) may suggest its involvement in alternative splicing and altered expression occurring during cellular transformation.40 1938


Eukaryotic translation initiation factor 5A-1 (eIF5A-1) is essential for proliferation of eukaryotic cells and can act as a biomarker for proliferation in intraepithelial neoplasia.41 Decreased levels of active eIF5A have been associated with inhibition of cell growth in murine mammary carcinoma cells,42 and growing evidence suggests a role for eIF5A in apoptosis by its ability to directly regulate p53.43 Taylor and co-workers44 reported overexpression-mediated proapoptotic activity of eIF5A, but unfortunately, eIF5A isoforms were not discriminated and comparison with results from this study are not possible. Proapoptotic effects of eIF5A were studied by Jin et al.45 in leukemic cells, and proteasomal-dependent regulation was reported. Herein, NT-3/TrkC-activation-dependent doublefold decreased eIF5A-1 levels are revealed. The isoform determined herein was A2 acetylated, and so far simply blockade of the N-terminal alanine was reported (http://www.expasy.org/uniprot/P63241). Again, aberrant stoichiometry of translation initiation complexes as well as binding of eIF5A to actively translating 80S ribosomes46 following TrkC activation may lead to altered translational activity. NT-3/TrkC activation affected levels of three chaperones: Heat-shock protein beta-1 (syn: Heat shock 27 kDa protein, Stress-responsive protein 27; HSP27) is observed in the mitotic spindle apparatus47 and is associated with cytoskeleton proteins actin and tubulin.48 HSP27 levels were decreased upon NT-3 stimulation of TrkC-transfected medulloblastoma cells from 24 h. Heat-shock 70 kDa protein 1 (syn.: HSP70.1, HSP70–1/HSP70–2, HSP70) levels were decreased at 48 h. No post-translational modifications were found, but chemical modifications of deamidation and oxidation were observed. It


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Figure 2. Continued

remains open, if deamidation of Q152 and Q175 in HSP27 and deamidation of Q330 and Q376 in HSP70 were results from deamidase activity or are technical in nature, and indeed, a receptor-modifying deamidase was detected in bacteria49 (http://www.expasy.org/uniprot/P08107). HSP27 and HSP70 appear to be involved in intracellular signaling, drug resistance, and thermotolerance of cancer

cells.50,51 Their concomitant increased expression protects against apoptosis52 and has been related to shorter diseasefree survival periods in breast cancer,51 esophageal squamous cell carcinoma,53 nonsmall cell lung cancer,54 and hepatocellular carcinoma55 patients. HSP27 and HSP70 expression is correlated with a better response to anticancer treatments: high HSP27 expression was related to favorable outcome in neuroJournal of Proteome Research • Vol. 7, No. 5, 2008 1939

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Figure 2. Continued

blastoma,56 whereas HSP70 overexpression was shown to predict a better response to chemotherapy in osteosarcomas.50 Findings of decreased heat shock protein levels are challenging studies on chemoresistance in TrkC-activated versus emptyvector control DAOY cells. T-complex protein 1 subunit gamma (syn: TCP-1-gamma, CCT-gamma; TCP-1) was decreased at earlier time points (from 1940


6 to 12 h). It assists folding of proteins upon ATP hydrolysis and chaperones actin and tubulin by guiding actin to its native conformation.57TCP-1 is known to play a role in early embryonic development.58 TCP-1 has been associated with dedifferentiation and progression of hepatocellular carcinoma,59,60 and expression is elevated in ovarian cancer.61

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Figure 2. Time course of all 12 proteins (represented by 14 spots) showing differential expression levels following TrkC/NT-3 activation. DAOY-TrkC cells represented by dark line; DAOY-empty vector-group represented by light line. Circles indicating individual expression levels of single gels. Table 2. Statistical Analysis and Expression Levels of NT-3/TrkC-Dependent Proteins Involved in Synthesis, Chaperoning, and Metabolism of Proteinsa protein name

UniProtKB accession no.

Cathepsin D U2 (RNU2) small nuclear RNA auxiliary factor 2, isoform b

P07339 Q96HC5

Heterogeneous nuclear ribonucleoprotein K Probable rRNA processing protein EBP2 26S proteasome non-ATPase regulatory subunit 13

P61978

40S ribosomal protein SA

Heterogeneous nuclear ribonucleoprotein H

significant time points

maximum fold change (time point)

Up-Regulated in DAOY-TrkC 1. Early (+/- transient) 6, 12 h +2-fold (6 h) 0–12 h +2.2-fold (6 h)

2. Late (+/- transient) 48 h +1.44-fold (48 h)

p-values

FDR

0.008 0.0008

0.05 0.01

0.012

0.15

Q99848

24, 48 h

+1.5-fold (24 h)

0.003

0.03

Q9UNM6

0–24 h

+1.8-fold (12 h)

0.0007

0.01

P08865

3. Sustained 6–48 h

+3.9-fold (24 h)

5.5 × 10–8

6.2 × 10–6

P31943

6–48 h 6–48 h 6, 24, 48 h

+3.9-fold (24 h) +2.9-fold (24 h) +2.8-fold (24 h)

0.0006 0.0007 0.0007

0.01 0.01 0.1

0.003

0.03

-1.9-fold (0 h)

0.0005

0.01

2. Late (+/- transient) 0–24 h -2.2-fold (0 h)

0.0003

0.007

T-complex protein 1, gamma subunit Heat-shock protein beta-1

P49368

Eukaryotic translation initiation factor 4H Eukaryotic translation initiation factor 5A Heat shock 70 kDa protein 1

Q15056

P04792

Down-Regulated in DAOY-TrkC 1. Early (+/- transient) 6, 12 h -2-fold (6 h) 0–12 h

P63241

48 h

-2.2-fold (48 h)

0.0008

0.026

P08107

0, 6, 24 h

-1.8-fold (0 h)

0.0007

0.01

a Fold changes are ratios of mean protein levels of individual spots from TrkC-transfected versus empty vector controls. p-Values refer to tests for group by time interaction in repeated measured analysis. FDR is the false-discovery rate.


research articles With respect to molecules involved in protein degradation, two proteins were up-regulated in TrkC-activated cells: 26S proteasome non-ATPase regulatory subunit 13 and cathepsin D. 26S proteasome non-ATPase regulatory subunit 13 is, as a part of the proteasome, responsible for highly selective intracellular protein degradation. While it is well-accepted that alterations in the exact timely degradation of proteins involved in growth control, apoptosis, signaling, and differentation contribute to carcinogenesis,62 no link to tumor biology has yet been described for this specific protein. The lysosomal aspartic protease cathepsin D, by contrast, is a key mediator of induced apoptosis with its proteolytic activity being generally involved in this event. Overexpression in breast cancer cells has been correlated with poor prognosis and the incidence of clinical metastasis. Several reports have indicated that cathepsin D stimulates cancer cell proliferation, increases metastatic potential, and stimulates tumor angiogenesis, as well as the development of drug resistance.63–65 Recent reports additionally state a dual function of cathepsin D in apoptosis, as it can strongly enhance chemo-sensitivity and apoptotic response to etoposide in cancer cells.66

Conclusion This study is the first to our knowledge to relate regulation of the protein machinery to TrkC activation and thereby to prognostic outcome in medulloblastoma. Although probably not reflecting the in vivo situation, cell lines are suitable tools for studying the response to external stimuli against an otherwise homogeneous genetic background. Although early signal transduction processes of Trk receptors have been analyzed, late effector proteins have not yet been described. This report presents proteins involved in protein synthesis, chaperoning, and metabolism being regulated by TrkC activation possibly associated with the biological changes induced by TrkC receptor activation in medulloblastoma. Analyses of individual functional effects of the respective effector proteins are underway and may reveal more detailed insights into the underlying causes for the better outcome of TrkC expressing medulloblastoma.

Acknowledgment. The excellent technical contribution of Julius Paul Pradeep John, Maureen Cabatic, Jae-Won Yang, Joo-Ho Shin, Leila Afjehi-Sadat, and Martin Murer is highly appreciated. We acknowledge the contribution of the CCRI, St. Anna Kinderspital, and “Forschungsgesellschaft fuer cerebrale Tumoren”. Supporting Information Available: Supplemental Figure 1, sample preparation, handling, and analysis. DAOYTrkC and DAOY-empty vector cells were stimulated with NT3. Control and treated cells were harvested at the different time points after neurotrophin addition and lysed, and equal amounts of protein were run on 2-DE gels. Experiments were repeated five times to adjust for interexperimental variations before analysis and quantification. Supplemental Figure 2, sequence alignment of U2 small nuclear RNA auxiliary factor 2 to Splicing factor U2AF 65 kDa subunit showing high identity (82%) (http://www.expasy.org/tools/blast/?Q96HC5). This material is available free of charge via the Internet at http:// pubs.acs.org. 1942



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PR700724A

Synthesis, Chaperoning, and Metabolism of Proteins Are Regulated

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