Mass Spectrometric Analysis of High-Mobility Group Proteins and

Feb 8, 2007 - Overexpression of HMGA1 proteins has been associated with almost ... sites of PTMs of HMGA1 proteins isolated from cancerous/normal ...
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Mass Spectrometric Analysis of High-Mobility Group Proteins and Their Post-Translational Modifications in Normal and Cancerous Human Breast Tissues Yan Zou and Yinsheng Wang* Department of Chemistry-027, University of California at Riverside, Riverside, California 92521-0403 Received February 8, 2007

High-mobility group (HMG) A1 proteins including HMGA1a and HMGA1b are chromosomal proteins that function in a variety of cellular processes such as cell growth, transcription regulation, neoplastic transformation, and progression. Overexpression of HMGA1 proteins has been associated with almost every type of cancer cells. Post-translational modifications (PTMs) of HMGA1 proteins in different types of human cancer cell lines have been extensively explored over the past decade. Here, we extended the identification of PTMs of HMGA1 proteins to human breast tumor tissue specimens with different carcinoma progression stages (metastatic and primary cancer) as well as the paired adjacent normal breast tissues. In this regard, we employed tandem mass spectrometry to examine the nature and sites of PTMs of HMGA1 proteins isolated from cancerous/normal human breast tissues. Novel PTMs of HMGA1a protein, that is, monomethylation at Lys30 and Lys54 as well as monophosphorylation at Ser43 and Ser48, were detected in cancer tissues. In these cancer tissues, we also found C-terminal constitutive phosphorylation in HMGA1a and HMGA1b as well as mono- and dimethylation of Arg25 in HMGA1a, which were previously found to be present in these proteins isolated from human cancer cell lines. Furthermore, a more complex spectrum of PTMs on HMGA1 proteins was correlated with a more aggressive malignancy in human breast cancer tissues. Keywords: high-mobility group proteins • post-translational modifications • breast cancer • metastasis • mass spectrometry

Introduction High-mobility group (HMG) proteins are a class of lowmolecular-weight nonhistone nuclear proteins that are composed of three subfamilies, HMGA (previously known as HMGI/Y), HMGB (formerly HMG-1/2), and HMGN (formerly HMG14/17).1 These proteins can all bind to DNA, have characteristic functional sequence motifs, and function as architectural transcription factors that are involved in a wide variety of cellular processes.2-4 The distinct feature of the HMGA proteins is the presence of three highly conserved independent “AThook” domains that preferentially bind to the minor groove of AT-rich DNA stretches.5-7 The human HMGA subfamily consists of three related members, namely, HMGA1a, HMGA1b, and HMGA2. HMGA1a and HMGA1b proteins result from the translation of splicing variants of a single gene (HMGA1) and differ only in that HMGA1b protein has an 11-residue internal deletion, whereas HMGA2 is coded by a separate gene.8,9 HMGA proteins play an important role in cell growth, chromatin remodeling, transcription regulation, differentiation, neoplastic transformation, and apoptosis.2-4,10 The expression of HMGA proteins is maximal during embryonic development and is low, or undetectable, in fully differentiated and nondi* Address correspondence to Yinsheng Wang; [email protected]; fax, (951) 827-4713; tel., (951) 827-2700.

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viding adult cells.3 More importantly, after embryogenesis, HMGA proteins are again expressed at high levels in transformed cells and tumors.10 Overexpression of HMGA protein has been noted in a large number of different cancer types, which include, among others,3 breast,11,12 prostate,13,14 colon,15,16 liver,17 lung,18 and thyroid19 tumor tissues. In vivo experiments with transgenic mice overexpressing different HMGA protein isoforms have confirmed that these proteins are oncogenic and promote tumor progression and metastasis to varying extents.3,10 The elevated levels of HMGA proteins have been suggested as good diagnostic markers for both neoplastic transformation and increased metastatic potential.3 It has also been implied that new therapies for cancer treatment or prevention based on the HMGA proteins may be feasible.3 HMGA1 proteins (HMGA1a and HMGA1b) are subjected to a variety of post-translational modifications (PTMs) in vivo and in vitro including phosphorylation, methylation, acetylation, and ADP-ribosylation.20-26 Prior studies have shown that HMGA1 proteins are substrates for various protein kinases, which result in the phosphorylation of different functional domains of the proteins. Protein kinase CK2 can catalyze the constitutive phosphorylations of Ser98, Ser101, and Ser102 in HMGA1a protein in vitro and in vivo.27-30 The function of this constitutive phosphorylation is not clearly understood, though its indirect effect on altering the protein’s DNA binding 10.1021/pr070072q CCC: $37.00

 2007 American Chemical Society

PTMs of HMGA1 Proteins in Tissues

property was demonstrated.10,20 Thr52 and Thr77 in HMGA1a could be phosphorylated by cdc2 kinase in vitro,31 and these two sites were also found to be phosphorylated in vivo in a cell cycle-dependent manner.31,32 In HMGA1a, Thr52 and Thr77 are on the N-termini of the second and third “AT hook” domains, respectively,3 and the phosphorylation of these two amino acids diminishes significantly (∼20-fold) the binding affinity of the proteins to AT-rich DNA.31,32 Furthermore, Thr20, Ser43, and Ser63 in HMGA1a were major sites of phosphorylation catalyzed by protein kinase C-R (PKCR).22 The PKCcatalyzed phosphorylation of HMGA1a again reduces the protein’s binding affinity toward DNA.33 Interestingly, a change in the degree of phosphorylation of HMGA1a has been demonstrated to occur during apoptosis, that is, hyperphosphorylation occurs at the early stage, whereas dephosphorylation takes place in the later stage of apoptosis.20 In addition to phosphorylation, methylation and acetylation of HMGA1 proteins have been reported recently.21,22,30,34-36 In this regard, Giancotti and co-workers21,34 showed by LC-MS that Arg25 in HMGA1a is monomethylated and the methylation is correlated with the execution of apoptosis in cancer cells. Our previous results by MALDI-MS/MS and LC-ESI-MS/MS illustrated that Arg25 in HMGA1a, but not HMGA1b, can be monomethylated as well as dimethylated symmetrically and asymmetrically in PC-3 human prostate cancer cells.30 Recent studies also revealed that three type-I PRMTs (protein arginine methyltransferases), that is, PRMT1, PRMT3, and PRMT6, could catalyze the methylation of HMGA1 proteins to different levels. In addition, several lysine residues in HMGA1a were found to be acetylated.24,36,37 In this context, Edberg et al.35 reported the distinct in vivo PTM patterns of HMGA1a protein in cultured breast cancer cells with differing metastatic potential. In that study, increased levels of acetylation and dimethylation of both lysine and arginine residues were found on HMGA1a protein from metastatic cells compared to the protein isolated from nonmetastatic cells.35 Although there have been many studies on the identification of the PTMs of HMGA1 proteins in different cell lines with various cancer types, under different stimuli, or with different stages of neoplastic transformation, it should be reminded that the cell types employed were permanent cell lines. These cells are immortalized, and they constitutively express elevated, or sometimes still very low, levels of detectable HMGA1 proteins compared to truly normal somatic cells.3 In the present study, we have targeted, for the first time, human breast tumor tissues, and have examined the PTMs of HMGA1a and HMGA1b proteins using tandem mass spectrometry, that is, LC-ESIMS/MS and MALDI-MS/MS. Four tumor/normal tissue pairs with different carcinoma progression stages were examined. Our results revealed that all HMG proteins were highly expressed in breast tumor tissues but at a very low level in adjacent normal tissues. In addition, we systematically examined the correlation of the PTMs of HMGA1 proteins with the metastatic potential of breast tumor.

Materials and Methods Tissues. Human breast tumor tissues and paired adjacent normal tissues were purchased from National Disease Research Interchange (NDRI, Philadelphia, PA). Tumor tissues with different carcinoma progression stages and normal tissues were obtained and examined in this study, including one metastatic cancer/normal (BM1202/BNM1207), two primary cancer/ normal (BC0213/BNC0222; BC0424/BNC0505) pairs, as well as

research articles one three-tissue set, that is, metastatic cancer/primary cancer/ normal tissues (BM1215/BC1213/BN1218). Tissues in each set/ pair were obtained from the same patient. All tissue samples were stored in a -80 °C freezer upon arrival in dry ice. Protein Extraction and Purification. Frozen tissue samples were thawed, minced with razor blades, and weighed to obtain 0.24-0.44 g of tissue prior to HMG protein extraction. In the presence of liquid nitrogen, tissue samples were grounded into fine powders and transferred into a 50-mL Beckman polyallomer centrifuge tube (Beckman Coulter, Inc., Fullerton, CA). The HMG proteins were extracted using 5% perchloric acid (PCA) according to previously described procedures.2,30,38 The same experimental procedures and conditions were applied for the protein extraction of all the examined tissue samples to achieve comparable extraction efficiency. The PCA-soluble proteins were purified by HPLC with a 2.1 × 150 mm C4 column (Grace Vydac) on an Agilent 1100 system (Agilent Technologies, Palo Alto, CA) monitored by absorbance detection at 220 nm. A 50-min gradient of 5-30% CH3CN in 0.1% TFA was employed, and the flow rate was 200 µL/min. Fractions containing HMG proteins were collected according to absorbance peaks or retention times in cases where protein peaks were not easily visible. The individual fractions were dried in a Speedvac concentrator and subjected to enzymatic digestion and mass spectrometric analysis. Trypsin Digestion. The dried HMG protein fractions were incubated with 0.1-0.3 µg of modified sequencing grade trypsin (Roche Applied Science, Indianapolis, IN) in a 50-mM NH4HCO3 solution (pH 8.0) at 37 °C overnight. The digestion mixtures were dried, desalted using C18 ZipTip (Millipore, Billerica, MA), and subjected to mass spectrometric analysis. Mass Spectrometry. Matrix-assisted laser desorption/ionization (MALDI)-MS and MS/MS measurements were performed on a QSTAR XL quadrupole/time-of-flight mass spectrometer equipped with an o-MALDI ion source (Applied Biosystems, Foster City, CA). The mass accuracy in MS/MS mode was approximately 10-30 ppm with external calibration. Tryptic peptides were dissolved in an aqueous solution of 0.1% TFA and mixed with an equal volume of matrix solution, consisting of a saturated solution of R-cyano-4-hydroxycinnamic acid in a solvent mixture of CH3CN, H2O, and TFA (50/ 50/0.1, v/v/v). On-line LC-electrospray ionization (ESI)-MS/MS was employed for peptide sequencing and PTM identification. A 0.50 × 150 mm Zorbax C18 capillary column (300 Å in pore size, 5 µm in particle size, Agilent Technologies) was used. The flow rate was 6 µL/min, and a 63-min gradient of 2-65% CH3CN in 0.6% aqueous solution of acetic acid was employed. A 2-µL aliquot was injected in each run, and the sample loop size was 10 µL. The effluent from the HPLC column was directed to an LTQ linear ion-trap mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA). Mass calibration for the LTQ mass spectrometer was carried out by standards provided by the instrument’s manufacturer, including caffeine, a tetrapeptide MRFA, and Ultramark 1621. The spray voltage was 4.0 kV, nitrogen was used as the sheath gas, and the capillary temperature was maintained at 275 °C. MS/MS was done in both data-dependent and preselected precursor-ion scan modes. The mass width for precursor ion isolation was 3 m/z units, and the collision gas was helium. The LC-MS/MS data were analyzed by a SEQUEST search of the Swiss-Prot protein database using BioWorks 3.2 software (Thermo Fisher Scientific, Inc., Waltham, MA). Monoisotopic mass was used for the Journal of Proteome Research • Vol. 6, No. 6, 2007 2305

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search, and the mass tolerances for peptide and fragment ions were set at 2.0 and 1.0 amu, respectively.

Results Differential Expression of HMG Proteins in Breast Tumor and Paired Adjacent Normal Tissues. We isolated HMG proteins from two metastatic human breast tumor and three primary tumor tissues as well as their adjacent normal tissues by using the acid extraction method.2,30,38 In this context, although other proteins can also be extracted by this method,39 the HMG proteins and histone H1 are the most abundant components in the extracts.2,39 The resulting extraction mixtures were separated by reversed-phase HPLC to purify each subfamily of the HMG proteins. The HPLC chromatograms of three human breast tumor/normal tissue pairs were shown in Figure 1, that is, BM1202/BNM1207, BC0213/BNC0222, and BC0424/ BNC0505. For tissue samples in each pair, the absorbance in the HPLC chromatograms were normalized to the same scale to better visualize the levels of HMG proteins in different tissue samples in the pair. In this respect, the corresponding weight of each tissue sample was also indicated in Figure 1. MALDI-MS analyses of the HMG proteins or trypsindigested peptides showed that the HMGN2, HMGN4, HMGN3a, HMGA1b, and HMGA1a proteins were eluted sequentially with the increase of acetonitrile content in the mobile phase (Figure 1). In addition, all HMG proteins are expressed in a much higher level in cancer tissues, that is, both metastatic and primary tumor tissues, than in the matching adjacent normal tissues. For example, the HPLC chromatogram in Figure 1A revealed that the HMG proteins obtained from 0.089 g of human breast metastatic cancer tissue (BM1202) were readily detected as obvious peaks. In contrast, with 0.094 g of adjacent normal tissue (BNM1207), only very low levels of HMGN2 and HMGN4 were detected. Other HMG proteins, especially HMGA1a and HMGA1b, were not detected as noticeable peaks in the chromatogram. Similarly, the HPLC chromatograms (not shown) of the metastatic cancer/primary cancer/normal tissue set (BM1215/BC1213/BN1218) also indicated a higher expression level of HMG proteins in cancerous tissues than that in the normal tissue. Identification of in Vivo PTMs of HMGA1a and HMGA1b Proteins in Cancerous/Normal Human Breast Tissues. We analyzed the HPLC fractions by MALDI-MS to identify the purified HMG proteins, especially HMGA1 proteins, extracted from human breast cancer tissue samples. In this respect, the MALDI mass spectrum of HMGA1a protein from metastatic cancer sample BM1202, that is, the 32.5-min fraction shown in Figure 1A, is depicted in Figure 2. The HMGA1a protein from BM1202 appears to be N-terminally acetylated (0P, m/z 11587, calculated m/z 11588 for the [M + H]+ ion), monophosphorylated (1P, m/z 11662, calculated m/z 11668 for the [M + H]+ ion), and diphosphorylated (2P, m/z 11747, calculated m/z 11748 for the [M + H]+ ion). In addition, the HMGA1a protein seems to be monomethylated (0P/1M, m/z 11599, calculated m/z 11602; 1P/1M, m/z 11680, calculated m/z 11682; 2P/1M, m/z 11760, calculated m/z 11762, Figure 2). The calculated m/z of HMGA1a was based on the amino acid sequence assuming that the N-terminus of the protein is acetylated.20-22 To establish unambiguously the sites of phosphorylation and methylation, we digested the HMGA1a protein with trypsin and subjected the resulting peptide mixture to MALDI-MS, MALDIMS/MS, and LC-ESI-MS/MS analyses. The LC-MS/MS results were further searched against the Swiss-Prot protein database 2306

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Figure 1. HPLC traces show the different expression levels of HMG proteins extracted from three pairs of human breast tumor and the paired tumor-adjacent normal tissues. (A) Metastatic tumor/normal tissue, BM1202 (bottom)/BNM1207 (top); (B) primary tumor/normal tissue, BC0213 (bottom)/BNC0222 (top); (C) primary tumor/normal tissue, BC0424 (bottom)/BNC0505 (top). The corresponding weights of each tissue sample that the HPLC traces were obtained from were indicated in parentheses.

by using BioWorks 3.2 software. As shown in Table 1, we identified the mono- and diphosphorylated peptide 88-106, mono- and dimethylated peptide 24-29, and monomethylated peptide 30-54. Our earlier study of HMGA1 proteins extracted from human prostate cancer cell line PC-3 had shown the constitutive phosphorylations of C-terminal peptide 88-106 at serine residues 98, 101, and 102, as well as the mono- and dimethylation of peptide 24-29 at Arg25.30 In the present study,

PTMs of HMGA1 Proteins in Tissues

Figure 2. MALDI-mass spectrum of the HMGA1a protein from metastatic human breast tumor tissue BM1202. (The fraction with a retention time of 32.5 min shown in Figure 1A.)

we have also observed corresponding modifications of HMGA1 proteins in human breast cancer tissues. To illustrate this, Figure 3 gave the product-ion spectra of the ESI-produced [M + 3H]3+ ions of the unmodified (the ion of m/z 746.3, Figure 3A), monophosphorylated (ion of m/z 773.0, Figure 3B), and diphosphorylated (ion of m/z 799.6, Figure 3C) forms of the peptide 88-106 (KLEKEEEEGISQESSEEEQ) in HMGA1a protein extracted from BM1202. In the monophosphorylated peptide segment (Figure 3B), we have established the phosphorylation site to be either Ser101 or Ser102. In this regard, the presence of unmodified b112+ and b122+ ions, together with the absence of monophosphorylated b11*2+ and b12*2+ (the symbol “*” represents the presence of a phosphorylated residue), suggests that Ser98 is not phosphorylated in this peptide. On the other hand, we observed monophosphorylated y7*, indicating that the phosphate is located on either Ser101 or Ser102. Other monophosphorylated fragment ions were also observed such as y9*, y11*, b16*2+, b17*2+, and so forth, supporting again the monophosphorylation of Ser101 or Ser102. However, we were not able to detect fragment ions arising from the cleavage at the amide bond between Ser101 and Ser102, that is, the y5 and b14 ions, in either the unmodified or the monophosphorylated form, which prevents us from determining whether the phosphorylation occurs exclusively on one of these two residues. In the diphosphorylated peptide segment, both Ser101 and Ser102 are phosphorylated, while Ser98 remains intact. As depicted in Figure 3C, the unmodified b112+, b122+, and b132+ ions are detected in the product-ion spectrum of unmodified peptide (Figure 3A), indicating again that Ser98 is not phosphorylated. Moreover, the presence of the phosphorylated fragment ions, y6** and y7**, demonstrates that both Ser101 and Ser102 are phosphorylated. Thus, we conclude that the Cterminal phosphorylation is heterogeneous; that is, Ser101, Ser102, or both can be phosphorylated. In addition to the phosphorylated peptides, MALDI-MS analysis of the tryptic digestion mixture of the HMGA1a protein from primary tumor sample BC0213 showed a mono- and dimethylated peptide segment containing amino acid residues 24-29 (GRGRPR, m/z 712.4 and 726.5, data not shown) in HMGA1a protein. To identify the site(s) of mono- and dimethylation of this peptide segment, we acquired the tandem mass spectra of the MALDI-produced ions of m/z 712.4 and m/z 726.5 (Figure 4). The tandem mass spectra closely resemble the results obtained for the same peptide segment of the

research articles HMGA1a protein from the PC-3 human prostate cancer cells.30 In this respect, we observed the “reporter neutral losses” for methylated arginines from precursor ion and product ions.30,40,41 In Figure 4A, the neutral losses of methylamine (CH3NH2, 31 Da) and monomethylcarbodiimide (CH3NdCdNH, 56 Da) support that this peptide bears a monomethylarginine (MMA). Additionally, we observed the unmodified y1 (m/z 175.1), y2 (m/z 272.2), and y3 (m/z 428.3) ions but not their methylated forms, supporting that neither Arg27 nor Arg29 is methylated. Thus, only Arg25 is monomethylated. Moreover, we found b4# (m/z 441.3), b5# + H2O (m/z 556.3), and product ions emanating from neutral losses from these fragments, for example, b4#-CH3NH2 (m/z 410.2), b4#-C(NH)2 (m/z 399.3), b5# + H2O-CH3NH2 (m/z 525.3), b5# + H2O-C(NH)2 (m/z 514.3), and b5# + H2OCH3NdCdNH (m/z 500.3) ions (Figure 4A, “#” designates those ions carrying a monomethylarginine, that is, a molecular mass increase of 14 Da). The observation of those fragment ions is also consistent with the monomethylation of Arg25. Similarly, the neutral loss of a dimethylamine [NH(CH3)2, 45 Da] indicates the presence of asymmetric NG,NG-dimethylarginine (aDMA) in this peptide ion (m/z 726.5, Figure 4B).30,40,41 On the other hand, the neutral losses of a methylamine (CH3NH2, 31 Da) and a dimethylcarbodiimide [C(NCH3)2, 70 Da] show that this peptide ion bears a symmetric NG,N′G-dimethylarginine (sDMA).30,40,41 Thus, MALDI-MS/MS results support that the two isomeric forms of dimethylarginine (DMA) are both present in this dimethylated peptide containing amino acids 24-29. In addition, we did not observe the neutral loss of a monomethylcarbodiimide (56 Da), which is a characteristic cleavage from monomethylated arginine (MMA), from either the precursor or fragment ions. Thus, we can exclude the possibility of the presence of two MMAs in this peptide. The site of dimethylation can again be determined from fragment ions arising from backbone cleavages. In this context, we observed unmodified y1 (m/z 175.1), y2 (m/z 272.2), and y3 (m/z 428.3) ions (Figure 4B), suggesting that neither Arg27 nor Arg29 is methylated. Furthermore, we found b4## (m/z 455.3), b4##CH3NH2 (m/z 424.3), b4##-C(NH)2 (m/z 413.3), b4##-C(NCH3)2 (m/z 385.2), b5## + H2O (m/z 570.4), b5## + H2O-CH3NH2 (m/z 539.3), b5## + H2O-NH(CH3)2 (m/z 525.3), and b5## + H2OC(NCH3)2 (m/z 500.3) ions. These results strongly support that the Arg25 is heterogeneously dimethylated; that is, both asymmetric and symmetric dimethylation are present in HMGA1a protein isolated from BC0213. From the metastatic breast tumor sample BM1202, we also detected a novel methylation on peptide segment 30-54 (K30QPPVSPGTALVGSQK45EPSEVPTPK54). The product-ion spectra of the ESI-produced [M + 4H]4+ ions of the unmodified (m/z 640.6) and monomethylated (m/z 644.4) peptide are shown in Figure 5, panels A and B, respectively. Comparison of the two tandem mass spectra revealed a 14-Da mass increase in many b and y ions in Figure 5B, indicating the presence of a methyl group in this peptide. Three lysine residues, that is, Lys30, Lys45, and Lys54, in this peptide can potentially be methylated. The presence of several fragment ions in both unmodified and monomethylated forms, such as b102+ (m/z 482.5) and b10#2+ (m/z 489.2), b112+ (m/z 538.8) and b11#2+ (m/z 545.9), b122+ (m/z 588.4) and b12#2+ (m/z 595.2), supports that Lys30 is partially monomethylated. Likewise, the coexistence of unmodified and monomethylated forms of y8 ions, that is, y82+ (m/z 427.9) and y8#2+ (m/z 434.9) and y8 (m/z 854.4) and y8# (m/z 868.5), allows us to conclude that Lys54 is partially methylated. In addition, other ion pairs such as y132+ (m/z 692.3) and y13#2+ (m/z 699.5), Journal of Proteome Research • Vol. 6, No. 6, 2007 2307

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Table 1. Summary of PTMs and Modified Peptides in HMGA1 Proteins Extracted from Cancerous and Normal Human Breast Tissuesa

a BM1202 and BM1215 are metastatic human breast tumor tissues; BC0213, BC0424, and BC1213 are primary breast tumor tissues; BN1218 is one of the normal human breast tissues examined in our study. b ND: not detectable. c Detected at a very low level in preselected precursor-ion mode. d The N-terminal methionine is not present in HMGA1a/A1b isolated from tissues, which is consistent with what was found for the two proteins isolated from cultured human cells.2 Shaded residues in HMGA1a/A1b sequences: AT-hook domains in HMGA1a/b proteins.

y142+ (m/z 742.2) and y14#2+ (m/z 749.0), b172+ (m/z 853.1) and b17#2+ (m/z 860.2) also support that both Lys30 and Lys54 are partially monomethylated. The MS/MS shown in Figure 5B, however, does not allow us establish unambiguously whether Lys45 is monomethylated. Other than C-terminal phosphorylation and methylation, we also detected a novel phosphorylation on the peptide segment with residues 30-54 (KQPPVSPGTALVGS43QKEPS48E VPTPK, MS/MS of the [M + 4H]4+ ion, of m/z 660.7, is shown in Figure 5C) of HMGA1a protein from the primary tumor sample BC0213. Interestingly, the monophosphorylation is again heterogeneously distributed. We have mapped the phosphate group to be heterogeneously distributed on Ser43 and Ser48. In this respect, we observed unmodified y4, b6, b10, b11, b12, and b13 ions, but not their phosphorylated forms, supporting that Ser35, Thr38, and Thr52 are unmodified. In addition, we detected several fragment ion pairs in both the unmodified and monophosphorylated forms, such as y82+ (m/z 427.9) and y8*2+ (m/z 467.7), y8 (m/z 854.5) and y8* (m/z 934.5), as well as b162+ (m/z 788.9) and b16*2+ (m/z 828.6), b172+ (m/z 853.4) and b17*2+ (m/z 893.4). These fragment ion pairs, together with the presence of y13*2+ (m/z 732.6) and y14*2+ (m/z 782.0) and the absence of the corresponding unmodified y132+ and y142+ ions, strongly suggest that Ser43 and Ser48 are both monophosphorylated to some extent in HMGA1a protein from the primary breast tumor sample BC0213. 2308

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Different PTM Patterns of HMGA1a and HMGA1b Proteins in Human Breast Cancer Tissues. Aside from the difference in PTMs sites of HMGA1a protein discussed above, we also examined the PTMs of HMGA1b protein extracted from the tissue samples tested above. In Table 1, we summarized all the modified peptides and the types and sites of PTMs in HMGA1a and HMGA1b proteins extracted from all breast tissues that we have examined. These results revealed different PTM patterns between HMGA1a and HMGA1b as well as among different tissue samples. Previous studies have led to the identification of the C-terminal constitutive phosphorylations on HMGA1a/b proteins27-30 as well as the mono- and dimethylation at Arg2521,30,34 on HMGA1a protein in various human cancer cell lines. Our results with human breast tumor tissues resembled previous results in cancer cell lines, indicating that these PTMs are distinctive modifications occurring in HMGA1 proteins in human cancer cells. In the present study, novel monomethylation and monophosphorylation of peptide segment 30-54 were identified in HMGA1a protein in mainly cancerous tissue samples, except that extremely low level of monomethylation of peptide 30-54 was observed using preselected precursor ion scan mode in one normal tissue BN1218 (Table 1). This peptide segment is present in HMGA1a, but not HMGA1b protein, and it links the first and second AT-hooks of HMGA1a (see protein sequences in Table 1). It is worth mentioning that Arg25 is not

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Figure 3. Product-ion spectra of the ESI-produced [M + 3H]3+ ion of (A) unmodified (m/z 746.3); (B) mono- (m/z 773.0); and (C) diphosphorylated (m/z 799.6) peptide 88-106 (KLEKEEEEGISQESSEEEQ) in HMGA1a protein isolated from metastatic human breast tumor tissue BM1202. Summary of observed fragment ions are shown at the bottom of panels B and C. An asterisk (*) indicates that an ion bears a phosphate group. “-P” and “-H2O” indicates the neutral loss of an H3PO4 and H2O, respectively.

methylated in HMGA1b protein isolated from any of the human breast tissue samples, which is consistent with the earlier results from studies with human cancer cell lines.21,30,34 Moreover, triphosporylation of C-terminal peptide 77-95 in

HMGA1b is observed in metastatic tissue sample BM1202, but triphosporylation of C-terminal peptide was not detected in HMGA1a protein in either cancer or normal tissue samples (Table 1). Journal of Proteome Research • Vol. 6, No. 6, 2007 2309

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Figure 4. Product-ion spectra of the MALDI-produced [M + H]+ ions of (A) mono- (m/z 712.3) and (B) dimethylated (m/z 726.5) HMGA1a peptide 24-29 (GRGRPR) from BC0213. Neutral losses are indicated in the spectrum. The symbol “#” designates fragment ions bearing a methyl group. Neutral loss from precursor ion or fragment ions are indicated in the spectra, i.e., the neutral losses of 17-, 31-, 42-, 45-, 56-, and 70-Da fragments correspond to the losses of an NH3, CH3NH2, C(NH)2, (CH3)2NH, CH3NdCdNH, and C(NCH3)2, respectively.

Along with the higher expression level of HMGA1a/b proteins found in all the cancerous tissue samples in contrast to that found in their adjacent normal tissues, we have also identified more PTMs in cancerous tissues than in normal tissues. We were able to detect the monomethylation of peptide 30-54 and monophosphorylation of peptide 88-106 in HMGA1a protein in human breast tissue BN1218, while both modified peptide ions are at a very low level as shown by LC-MS/MS data acquired in preselected precursor-ion scan mode (Table 1). No PTMs were found for HMGA1b protein extracted from normal human breast tissues, though the failure in observing these PTMs could also be attributed to the lower level of expression of this protein in normal tissues. Of the two malignancy stages of human breast tumor, that is, metastatic and primary tumor tissues, more PTMs were observed in metastatic than in primary tissues (Table 1). In this context, all discovered PTMs are present in metastatic cancer tissues, while the diphosphorylation of the C-terminal peptide in HMGA1a and HMGA1b and triphosphorylation of the same peptide segment in HMGA1b were not detectable in primary cancer tissues.

Discussion Most normal, differentiated mammalian cells express extremely low levels of HMGA1 gene products; the HMGA1 proteins are, however, overexpressed in a variety of human and animal cancers.42 HMGA1 proteins play a role in the transcription of many genes involved in different steps of the metastatic cascade and have been proposed to be diagnostic markers for 2310

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both neoplastic transformation and metastatic progression as well as potential therapeutic target for patients [reviewed in refs 3, 4, 10, and 42. In particular, the correlation between HMGA1 expression with malignancy and metastasis in breast cancer was reviewed in ref 42]. HMGA proteins can adopt a variety of PTMs that are dynamic, dependent on the progression of cell cycle and exposure to environmental stimuli, and capable of modulating their interactions with DNA and other proteins.3,4,10,42 However, all earlier studies on PTMs of HMGA1 proteins are based on proteins isolated from cultured human cell lines. In this context, it is important to investigate fresh or fresh-frozen tissue without fixation so that the important aspects of human proteome and their modifications can be preserved.43 In the present study, we have obtained two metastatic human breast tumor specimens, three primary tumor tissue samples, and the matching adjacent normal tissue samples. Systematic investigation of PTMs of HMGA1 proteins extracted from these human breast tissues was performed using mass spectrometric approaches, especially tandem MS, that is, on-line LC-ESI-MS/MS and MALDI-MS/MS. The constitutive phosphorylations of C-terminal acidic tails of HMGA1a and HMGA1b proteins have been detected in human cancer cell lines in our previous studies.30,36 HMGA1 proteins are direct downstream targets for a number of signal transduction pathways leading to the phosphorylation of specific amino acid residues.3,4 In particular, the phosphorylation on these three serine residues has been demonstrated

PTMs of HMGA1 Proteins in Tissues

research articles

Figure 5. Product-ion spectra of the ESI-produced [M + 4H]4+ ion of (A) unmodified (m/z 640.6); (B) monomethylated (m/z 644.4); and (C) monophosphorylated (m/z 660.7) peptide 30-54 (KQPPVSPGTALVGSQKEPSEVPTPK) in HMGA1a protein isolated from metastatic human breast tumor tissue BM1202 (A, B) and primary tumor tissue BC0213 (C). Summary of observed fragment ions are shown at the bottom of panels B and C. The symbols “u” and “#” designate unmodified fragment ions and fragment ions carrying a methyl group, respectively. An asterisk (*) indicates that an ion bears a phosphate group. “-P” and “-H2O” represent the neutral losses of an H3PO4 and H2O, respectively. Journal of Proteome Research • Vol. 6, No. 6, 2007 2311

research articles to be catalyzed by protein kinase CK2 and may play a role in the alteration of protein conformation and the modulation of HMGA1’s DNA-binding properties.10,30 In the present study, we have detected the same constitutive C-terminal phosphorylation in HMGA1a and HMGA1b proteins extracted from human breast tumor tissues. More interestingly, the phosphorylation levels in the more aggressive metastatic breast cancer tissues (Table 1) are higher than those in the less aggressive primary cancer tissues. In addition, higher expression levels of HMGA1 proteins were observed in metastatic (Figure 1) than in primary cancer tissue specimens, which was consistent with the previous results obtained by Dolde et al.11 with human breast cancer cell lines, indicating that the increased expression of HMGA1 proteins correlates with a more aggressive phenotype of malignancy. We also observed, for the first time, a heterogeneous monophosphorylation of HMGA1a protein on Ser43 and Ser48 in one metastatic cancer tissue (BM1215) and two primary cancer tissues (BC0213 and BC1213) (Table 1). Ser43 in HMGA1a has been previously reported to be a high affinity site for protein kinase C (PKC) in vivo and in vitro.22,33 However, Thr20 and Ser63, other PKC recognition sites in HMGA1a, were not phosphorylated in any tissue samples that we examined. A significant reduction of DNA-binding affinity was found for the HMGA1a protein phosphorylated by PKC.22,33 Moreover, we identified a novel phosphorylation site at Ser48 in HMGA1a, and the enzyme(s) catalyzing this phosphorylation has not been reported. Both Ser43 and Ser48 are located within the spanning region between the first and second DNA-binding domains (AT-hooks) of the HMGA1a protein and are in close proximity to the N-terminus of the second AT-hook. Thus, we speculate that the phosphorylation of these two sites may decrease the binding affinity of HMGA1a protein to chromosomal DNA by compromising the positive charges of the AThooks. It is worth noting that the peptide segment with residues 30-54, due to alternative splicing, is not present in the HMGA1b isoform. The monomethylation of Arg25 in the first AT-hook of HMGA1a from tumor cell lines has been demonstrated to be associated with hyperphosphorylation/ dephosphorylation processes in apoptosis.21,34 More recently, we reported that the same arginine residue is dimethylated, both symmetrically and asymmetrically, in PC-3 prostate cancer cells.30 In vitro methylation of HMGA1 proteins with several PRMTs was examined recently, and the results suggested that PRMT6 might be a good candidate enzyme for the in vivo methylation of HMGA1 proteins.44,45 PRMT6 methylates HMGA1a protein primarily in the second AT-hook at Arg57 and Arg59 in vitro; however, our recent study showed that PRMT1 methylates preferentially HMGA1 proteins at Arg25 (unpublished data). The methylation of Arg25 in HMGA1a has been suggested to increase steric hindrance and affect hydrogen bonding interaction(s).30,46 In addition, we identified novel lysine methylation in this protein. In this respect, we found heterogeneous monomethylation on Lys30 and Lys54 in two metastatic human breast tumor samples (BM1202 and BM1215) and one primary tumor tissue sample (BC1213, Table 1). This modification on peptide 3054 is also located in the linker region of the first and second AT-hooks of the HMGA1a protein (see protein sequence in Table 1) that contains the 11-amino acid deletion in HMGA1b isoform. The biological function(s) of this monomethylation is not clear. 2312

Journal of Proteome Research • Vol. 6, No. 6, 2007

Zou and Wang

Many studies have demonstrated the different in vivo PTM patterns of HMGA1a and A1b isoforms from different tissue culture cell types.22,30,47 Microarray analysis of transcription profiles showed that the two isoforms differentially regulate specific genes.48 Furthermore, overexpression of HMGA1b, rather than HMGA1a, promotes tumor formation and metastasis in nude mice.48 Our present study revealed different PTM patterns of HMGA1a and HMGA1b in human breast cancer tissues, which also supports the different biological functions of these two splicing variants. In addition to the overexpression of HMGA1 proteins as potential cancer markers for both neoplastic transformation and metastatic progression, the types and sites of PTMs of HMGA1 proteins may also serve as an important feature of cancer tissues. The findings discussed in the present study from human breast tumor/normal tissue specimens have demonstrated the different patterns of PTMs of HMGA1a and HMGA1b proteins that are associated with neoplastic malignancies. A more complex spectrum of PTMs of HMGA1 proteins from human breast cancer tissues was observed and correlated with a more aggressive malignant phenotype. This information may help elucidate the mechanism of metastasis. In this respect, the PTMs that we observed for metastatic tumor specimens may facilitate the identification of protein kinases and other enzymes (i.e., PRMTs and histone methyltransferases) that induce these biochemical modifications, which in turn may allow for the elucidation of signal transduction pathways in the metastasis of breast tumor. Along this line, protein kinases CR and C were previously shown to promote an aggressive metastatic breast cancer phenotype.49,50 Moreover, our results suggest that there are similarities and distinct differences of the PTMs of HMGA1 proteins between human breast cancer cell lines and tumor specimens, which again underscores the importance of direct analysis of the proteome and PTMs with human tissues.43 It is worth noting that, due to the difficulty in procuring these cancer and matched normal tissues, a relatively small number of tissue samples were employed in the current study. It is important to extend the study to a large number of tissue samples and to other types of human cancer tissues in the future. Abbreviations: HMG, high-mobility group; PTM, posttranslational modification; CK2, protein kinase CK2 (or casein kinase 2); CIP, calf intestinal alkaline phosphatase; PRMT, protein arginine methyltransferase; CARM, coactivator-associated arginine methyltransferase; MMA, NG-monomethylarginine; aDMA, asymmetric NG,NG-dimethylarginine; sDMA, symmetric NG,N′G-dimethylarginine; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption/ionization; TOF, timeof-flight; MS/MS, tandem mass spectrometry; TFA, trifluoroacetic acid; PCA, perchloric acid.

Acknowledgment. The authors thank the National Disease Research Interchange (NDRI) for supplying the breast tissue samples and Dr. Songqin Pan at the W. M. Keck Proteomics Laboratory at UC Riverside for assistance with MALDI-MS/MS measurements. This work was supported by the National Institutes of Health (R01 CA101864). References (1) Bustin, M.; Reeves, R. High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. Prog. Nucleic Acid Res. Mol. Biol. 1996, 54, 35-100. (2) Reeves, R. HMGA proteins: isolation, biochemical modifications, and nucleosome interactions. Methods Enzymol. 2004, 375, 297322.

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Zou and Wang (48) Reeves, R.; Edberg, D. D.; Li, Y. Architectural transcription factor HMGI(Y) promotes tumor progression and mesenchymal transition of human epithelial cells. Mol. Cell. Biol. 2001, 21, 57594. (49) Tan, M.; Li, P.; Sun, M.; Yin, G.; Yu, D. Upregulation and activation of PKCR by ErbB2 through Src promotes breast cancer cell invasion that can be blocked by combined treatment with PKCR and Src inhibitors. Oncogene 2006, 25, 3286-3295. (50) Pan, Q.; Bao, L. W.; Kleer, C. G.; Sabel, M. S.; Griffith, K. A.; Teknos, T. N.; Merajver, S. D. Protein kinase C is a predictive biomarker of aggressive breast cancer and a validated target for RNA interference anticancer therapy. Cancer Res. 2005, 65, 8366-8371.

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