HMGA1B/2 transcriptionally activated-POU1F1 facilitates gastric carcinoma metastasis via CXCL12/CXCR4 axis-mediated macrophage polarization.

Tumor-associated macrophages (TAMs) in the tumor microenvironment contribute to poor prognosis in gastric cancer (GC). However, the underlying mechanism by which TAMs promote GC progression and metastasis remains elusive. Expression of POU1F1 was detected in 60 matched GC-normal tissue pairs using qRT-PCR and immunohistochemistry (IHC) analysis. The correlation between POU1F1 and the clinical-pathological factors of GC patients were further assessed. Cell proliferation was monitored by CCK-8, colony formation, and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assays. Cell migration and invasion were assessed by transwell assays. The impact on angiogenesis was evaluated by tube formation assay. Xenograft model was generated to investigate the role of POU1F1 on tumor growth and lung metastasis in vivo. GST pull-down and Co-immunoprecipitation (Co-IP) were used to study the interaction between HMGA1B/2 and POU1F1. Chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were performed to investigate the transcriptional regulation of POU1F1. Flow cytometry was performed to detect the surface expression of macrophage markers. Upregulated POU1F1 observed both in GC tissues and cell lines was positively correlated with poor prognosis. Knockdown of POU1F1 inhibited cell proliferation, migration, invasion, and angiogenesis in vitro, and suppressed tumor growth in vivo. HMGA1B/2 transcriptionally activated-POU1F1. POU1F1 promoted GC progression via regulating macrophage proliferation, migration, polarization, and angiogenesis in a CXCL12/CXCR4-dependent manner. POU1F1 also promoted GC metastasis in lung by modulating macrophage polarization through CXCL12/CXCR4 axis in vivo. HMGA1B/2-upregulated POU1F1 promoted GC metastasis via regulating macrophage polarization in a CXCL12/CXCR4-dependent manner.

Role of High-mobility Group Protein A Isoforms and Their Clinicopathologic Significance in Primary Retinoblastoma.

BACKGROUND: High-mobility group proteins A (HMGA) are more abundant in rapidly dividing and transformed cells. These are a group of proteins regulating tumorigenesis and tumor invasion. Increased expression of HMGA1 and HMGA2 has been reported in various benign and malignant tumors. The aim of the present study was to analyze expression of HMGA1 and HMGA2 proteins in retinoblastoma. METHODS: Protein expression of HMGA1 and HMGA2 in 80 formalin-fixed retinoblastoma tissues was performed by immunohistochemistry, and their mRNA expressions were analyzed on 40 fresh primary enucleated retinoblastoma samples by semiquantitative reverse transcription polymerase chain reaction. Results were then correlated with clinicopathologic parameters. RESULTS: Immunohistochemical analysis of HMGA1 and HMGA2 was seen in 56.25% and 58.75% of retinoblastoma cases, respectively. mRNA expressions of HMGA1 and HMGA2 was found to be 57.55% and 62.5%, respectively. The mRNA results correlated well with immunostaining results. Expression of both HMGA1 and HMGA2 was significantly associated with choroidal invasion and poor tumor differentiation. CONCLUSIONS: HMGA1 and HMGA2 proteins may contribute to tumorigenesis of Rb. Expression of HMGA1 and HMGA2 predicts poor prognosis and could serve as a therapeutic target in the management of RB. Further experiments are needed to determine the role of these proteins as therapeutic targets in tumorigenesis.

HMGA1-pseudogenes and cancer.

Pseudogenes are DNA sequences with high homology to the corresponding functional gene, but, because of the accumulation of various mutations, they have lost their initial functions to code for proteins. Consequently, pseudogenes have been considered until few years ago dysfunctional relatives of the corresponding ancestral genes, and then useless in the course of genome evolution. However, several studies have recently established that pseudogenes are owners of key biological functions. Indeed, some pseudogenes control the expression of functional genes by competitively binding to the miRNAs, some of them generate small interference RNAs to negatively modulate the expression of functional genes, and some of them even encode functional mutated proteins. Here, we concentrate our attention on the pseudogenes of the HMGA1 gene, that codes for the HMGA1a and HMGA1b proteins having a critical role in development and cancer progression. In this review, we analyze the family of HMGA1 pseudogenes through three aspects: classification, characterization, and their possible function and involvement in cancer.

C-terminal extension of calmodulin-like 3 protein from Oryza sativa L.: interaction with a high mobility group target protein.

A large number of calmodulin-like (CML) proteins are present in plants, but there is little detailed information on the functions of these proteins in rice (Oryza sativa L.). Here, the CML3 protein from rice (OsCML3) and its truncated form lacking the C-terminal extension (OsCML3m) were found to exhibit a Ca2+-binding property and subsequent conformational change, but the ability to bind the CaM kinase II peptide was only observed for OsCML3m. Changes in their secondary structure upon Ca2+-binding measured by circular dichroism revealed that OsCML3m had a higher helical content than OsCML3. Moreover, OsCML3 was mainly localized in the plasma membrane, whereas OsCML3m was found in the nucleus. The rice high mobility group B1 (OsHMGB1) protein was identified as one of the putative OsCML3 target proteins. Bimolecular fluorescence complementation analysis revealed that OsHMGB1 bound OsCML3, OsCML3m or OsCML3s (cysteine to serine mutation at the prenylation site) in the nucleus presumably through the methionine and phenylalanine-rich hydrophobic patches, confirming that OsHMGB1 is a target protein in planta. The effect of OsCML3 or OsCML3m on the DNA-binding ability of OsHMGB1 was measured using an electrophoretic mobility shift assay. OsCML3m decreased the level of OsHMGB1 binding to pUC19 double-stranded DNA whereas OsCML3 did not. Taken together, OsCML3 probably provides a mechanism for manipulating the DNA-binding ability of OsHMGB1 in the nucleus and its C-terminal extension provides an intracellular Ca2+ regulatory switch.

CBX7 and HMGA1b proteins act in opposite way on the regulation of the SPP1 gene expression.

Several recent studies have reported the Polycomb Repressive Complex 1 member CBX7 as a tumor-suppressor gene whose expression progressively decreases in different human carcinomas in relation with tumor grade, malignant stage and poor prognosis. We have previously demonstrated that CBX7 is able to inhibit the expression of the SPP1 gene, encoding the chemokine osteopontin that is over-expressed in cancer and has a critical role in cancer progression. Here, we have analyzed the mechanism by which CBX7 regulates the SPP1 gene expression. We show that the SPP1 transcriptional regulation mechanism involves the CBX7-interacting protein HMGA1b, that acts as a positive regulator of the SPP1 gene. In fact, we demonstrate that, in contrast with the transcriptional activity of CBX7, HMGA1b is able to increase the SPP1 expression by inducing the activity of its promoter. Moreover, we show that CBX7 interferes with HMGA1b on the SPP1 promoter and counteracts the positive transcriptional activity of HMGA1b on the SPP1 expression. Furthermore, since we found that also the NF-κB complex resulted involved in the modulation of the SPP1 expression in thyroid cells, we suppose that CBX7/HMGA1b/NF-κB could take part in the same transcriptional mechanism that finally leads to the regulation of the SPP1 gene expression. Taken together, our data show the important role played by CBX7 in the negative regulation of the SPP1 gene expression, thus contributing to prevent the acquisition of a malignant phenotype.

HMGA1 and HMGA2 expression and comparative analyses of HMGA2, Lin28 and let-7 miRNAs in oral squamous cell carcinoma.

BACKGROUND: Humans and dogs are affected by squamous cell carcinomas of the oral cavity (OSCC) in a considerably high frequency. The high mobility group A2 (HMGA2) protein was found to be highly expressed in human OSCC and its expression was suggested to act as a useful predictive and prognostic tool in clinical management of oral carcinomas. Herein the expression of HMGA2 and its sister gene HMGA1 were analysed within human and canine OSCC samples. Additionally, the HMGA negatively regulating miRNAs of the let-7 family as well as the let-7 regulating gene Lin28 were also comparatively analysed. Deregulations of either one of these members could affect the progression of human and canine OSCC. METHODS: Expression levels of HMGA1, HMGA2, Lin28, let-7a and mir-98 were analysed via relative qPCR in primary human and canine OSCC, thereof derived cell lines and non-neoplastic samples. Additionally, comparative HMGA2 protein expression was analysed by immunohistochemistry. RESULTS: In both species, a significant up-regulation of the HMGA2 gene was found within the neoplastic samples while HMGA1 expression did not show significant deregulations. Comparative analyses showed down-regulation of mir-98 in human samples and up-regulation of let-7a and mir-98 in canine neoplastic samples. HMGA2 immunostainings showed higher intensities within the invasive front of the tumours than in the centre of the tumour in both species. CONCLUSIONS: HMGA2 could potentially serve as tumour marker in both species while HMGA1 might play a minor role in OSCC progression. Comparative studies indicate an inverse correlation of HMGA2 and mir-98 expression in human samples whereas in dogs no such characteristic could be found.

Expression of high mobility group A proteins in oral leukoplakia.

BACKGROUND: Oral leukoplakia (LPL) is considered a potentially malignant disorder in the oral cavity and the gastric tract. High mobility group A (HMGA) proteins are important in the transformation of normal cells into cancer cells, but there is a lack of knowledge on their importance in oral cancer development. The aim of the current project was to investigate HMGA expression in LPLs with different levels of dysplasia. MATERIALS AND METHODS: Biopsies were histologically processed to visualize the expression of HMGA1 and HMGA2 using immunohistochemistry. RESULTS: An increase of HMGA1-positive cells correlating to the degree of dysplasia was registered in the epithelium and in the connective tissue. HMGA2 expression was seen in the epithelium and in the connective tissue but with no obvious correlation to the level of dysplasia. CONCLUSION: This is, to our knowledge, the first study showing the expression of HMGA proteins in healthy and non-healthy oral mucosa.

The Wnt/ß-catenin/T-cell factor 4 pathway up-regulates high-mobility group A1 expression in colon cancer.

High-mobility group A1 (HMGA1) encodes proteins that act as mediators in viral integration, modification of chromatin structure, neoplastic transformation and metastatic progression. Because HMGA1 is overexpressed in most cancers and has transcriptional relationships with several Wnt-responsive genes, we explored the involvement of HMGA1 in Wnt/ß-catenin/TCF-4 signalling. In adenomatous polyposis coli (APC(Min/+)) mice, we observed significant up-regulation of HMGA1 mRNA and protein in intestinal tumours when compared with normal intestinal mucosa. Conversely, restoration of Wnt signalling by the zinc induction of wild-type APC resulted in HMGA1 down-regulation in HT-29 cells. Because APC mutations are associated with mobilization of the ß-catenin/TCF-4 transcriptional complex and subsequent activation of downstream oncogenic targets, we analyzed the 5'-flanking sequence of HMGA1 for putative TCF-4 binding elements. We identified two regions that specifically bind the ß-catenin/TCF-4 complex in vitro and in vivo, identifying HMGA1 as an immediate target of the ß-catenin/TCF-4 signalling pathway in colon cancer. Collectively, these findings strongly implicate Wnt/ß-catenin/TCF-4 signalling in regulating HMGA1 to further expand the extensive regulatory network affected by Wnt/ß-catenin/TCF-4 signalling.

Interaction of adriamycin with a promoter region of hmga1 and its inhibitory effect on HMGA1 expression in A431 human squamous carcinoma cell line.

The high mobility group A1 (HMGA1) are non-histone chromosomal proteins consisting of HMGA1a and HMGA1b which act as architectural transcription factors. Elevated levels of HMGA1 are reported in a number of human cancers and suggested as tumor markers. Due to their role in neoplastic transformation and tumor progression, we considered HMGA1 as a potential target for downregulation at the transcriptional level. The present paper deals with the binding of a widely used chemotherapeutic agent, adriamycin (ADM), to hmg a1 gene promoter (-304 to -284), 21RY, and its effect on the expression of hmga1 at the mRNA and protein levels and further its cytotoxic efficacy in A431 cells. A strong complex (21RY-ADM) formation caused hypochromic and bathochromic changes in UV-absorption, considerable spectral changes in circular dichroism of adriamycin and DNA and significant quenching of fluorescence emission of ADM. Thermodynamics of 21RY-ADM interaction was studied by isothermal titration and differential scanning calorimetric techniques that revealed the binding to be exothermic and favored by both negative enthalpy and positive entropy changes. Further, even low concentrations (10.3 nM) of ADM showed cytotoxicity on human squamous carcinoma cells (A431) and caused downregulation (by ∼ 70%) of hmga1 at mRNA and protein levels. Present findings clearly support the inhibitory effect of ADM on hmg a1 which quite probably is the consequence of its binding to the targeted region of hmg a1. Therefore, it appears that hmg a1 is a novel potential chemotherapeutic target in treating carcinomas of epithelial origin like prostate, breast, thyroid etc.

Expression of a truncated Hmga1b gene induces gigantism, lipomatosis and B-cell lymphomas in mice.

HMGA1 gene rearrangements have been frequently described in human lipomas. In vitro studies suggest that HMGA1 proteins have a negative role in the control of adipocyte cell growth, and that HMGA1 gene truncation acts in a dominant-negative fashion. Therefore, to define better the role of the HMGA1 alterations in the generation of human lipomas, we generated mice carrying an Hmga1b truncated (Hmga1b/T) gene. These mice develop a giant phenotype together with a drastic expansion of the retroperitoneal and subcutaneous white adipose tissue. We show that the activation of the E2F pathway likely accounts, at least in part, for this phenotype. Interestingly, the Hmga1b/T mice also develop B-cell lymphomas similar to that occurring in Hmga1-knockout mice, supporting a dominant-negative role of the Hmga1b/T mutant also in vivo.

HMGA2: A pituitary tumour subtype-specific oncogene?

The high mobility group AT-hook (HMGA) proteins, a family of DNA architectural factors, are highly expressed during embryogenesis and play a crucial role in several different biological processes, as well as in tumorigenesis of a wide range of tissues, including pituitary. Indeed, HMGA2 has been found rearranged and amplified in human prolactinomas, and transgenic mice overexpressing either Hmga1 or Hmga2 develop pituitary adenomas secreting prolactin and growth hormone. Here, we overview HMGA proteins in human tumours, focusing on pituitary adenomas and the mechanisms by which the HMGA proteins are involved in their onset and development. Different HMGA-dependent potential drives of pituitary oncogenesis are discussed as future research directions in the field.

Two new miR-16 targets: caprin-1 and HMGA1, proteins implicated in cell proliferation.

BACKGROUND INFORMATION: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16. RESULTS: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. CONCLUSIONS: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.

Homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and modulates its DNA binding affinity.

The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook.

A quantitative study on the in vitro and in vivo acetylation of high mobility group A1 proteins.

High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64 approximately Lys-70 > Lys-66 > Lys-14 approximately Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70 approximately Lys-73 > Lys-64 approximately Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.

A mass spectrometric study on the in vitro methylation of HMGA1a and HMGA1b proteins by PRMTs: methylation specificity, the effect of binding to AT-rich duplex DNA, and the effect of C-terminal phosphorylation.

HMGA1a and HMGA1b are members of one subfamily of non-histone chromosomal high-mobility group (HMG) proteins. They bind to various DNA-related substrates, including the minor groove of AT-rich duplex DNA sequences, and have been postulated to be architectural transcription factors functioning in a wide variety of cellular processes. Post-translational modifications of HMGA1 proteins, such as phosphorylation, acetylation, and methylation, are widely observed in tumor cells in vivo and correlated with the modulation of protein function. Here, we investigated the in vitro methylation of recombinant human HMGA1a and HMGA1b proteins by three members of the protein arginine methyltransferase (PRMT) family: PRMT1, PRMT3, and PRMT6. PRMT1 and PRMT3 showed a preference for methylating arginine residues in the first AT-hook of HMGA1 proteins, whereas PRMT6 methylated mainly residues in the second AT-hook. The initial sites of methylation catalyzed by PRMT1 and PRMT3 were mapped by tandem mass spectrometry to be Arg25 and Arg23, respectively, while we confirmed that the initial sites of methylation catalyzed by PRMT6 were at Arg57 and Arg59. Our results also revealed that binding of HMGA1 proteins to AT-rich duplex DNA, but not GC-rich duplex DNA, significantly inhibited the methylation efficiency of all of the PRMTs toward HMGA1 proteins. Moreover, C-terminal constitutive phosphorylation of HMGA1 proteins induced by protein kinase CK2 did not have any appreciable effect on the in vitro methylation of HMGA1. Our results suggest that PRMT1 might be involved in the previously reported methylation of Arg25 in HMGA1a in vivo.

Mass spectrometric analysis of high-mobility group proteins and their post-translational modifications in normal and cancerous human breast tissues.

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.

Dual role for SUMO E2 conjugase Ubc9 in modulating the transforming and growth-promoting properties of the HMGA1b architectural transcription factor.

Members of the HMGA1 (high mobility group A1) family of architectural transcription factors, HMGA1a and HMGA1b, play important roles in many normal cellular processes and in tumorigenesis. We performed a yeast two-hybrid screen for HMGA1-interacting proteins and identified the SUMO E2 conjugase Ubc9 as one such partner. The Ubc9-interacting domain of HMGA1 is bipartite, consisting of a proline-rich region near the N terminus and an acidic domain at the extreme C terminus, whereas the HMGA1-interacting domain of Ubc9 comprises a single region previously shown to associate with and SUMOylate other transcription factors. Consistent with these findings, endogenous HMGA1 proteins and Ubc9 could be co-immunoprecipitated from several human cell lines. Studies with HMGA1b proteins containing mutations of either or both Ubc9-interacting domains and with Ubc9-depleted cell lines indicated that the proline-rich domain of HMGA1b positively influences transformation and growth, whereas the acidic domain negatively influences these properties. None of the changes in HMGA1 protein functions mediated by Ubc9 appears to require SUMOylation. These findings are consistent with the idea that Ubc9 can act as both a positive and negative regulator of proliferation and transformation via its non-SUMO-dependent interaction with HMGA1 proteins.

High Mobility Group A1 (HMGA1) proteins interact with p53 and inhibit its apoptotic activity.

HMGA gene overexpression and rearrangements are frequent in several tumours, but their oncogenic function is still unclear. Here we report of a physical and functional interaction between High Mobility Group A1 (HMGA1) protein and p53 oncosuppressor. We found that HMGA1 binds p53 in vitro and in vivo, and both proteins are present in the same complexes bound to the Bax gene promoter. HMGA1 interferes with the p53-mediated transcription of p53 effectors Bax and p21(waf1) while cooperates with p53 in the transcriptional activation of the p53 inhibitor mdm2. This transcriptional modulation is associated with a reduced p53-dependent apoptosis in cells expressing exogenous HMGA1 and p53, or in cells expressing endogenously the proteins and in which p53 was activated by UV-irradiation. Furthermore, antisense inhibition of HMGA1b expression dramatically increases the UV-induced p53-mediated apoptosis. These data define a new physical and functional interaction between HMGA1 and p53 that modulates transcription of p53 target genes and inhibits apoptosis.

Tandem mass spectrometry for the examination of the posttranslational modifications of high-mobility group A1 proteins: symmetric and asymmetric dimethylation of Arg25 in HMGA1a protein.

High-mobility group (HMG) A1a and A1b proteins are among a family of HMGA proteins that bind to the minor groove of AT-rich regions of DNA. Here we employed tandem mass spectrometry and determined without ambiguity the sites of phosphorylation and the nature of methylation of HMGA1 proteins that were isolated from the PC-3 human prostate cancer cells. We showed by LC-MS/MS that Ser101 and Ser102 were completely phosphorylated in HMGA1a protein, whereas only a portion of the protein was phosphorylated at Ser98. We also found that the HMGA1b protein was phosphorylated at the corresponding sites, that is, Ser90, Ser91 and Ser87. In addition, Arg25, which is within the first DNA-binding AT-hook domain of HMGA1a, was both mono- and dimethylated. Moreover, both symmetric and asymmetric dimethylations were observed. The closely related HMGA1b protein, however, was not methylated. The unambiguous identification of the sites of phosphorylation and the nature of methylation facilitates the future examination of the biological implications of the HMGA1 proteins.