Down-regulation of B cell receptor signaling by hematopoietic progenitor kinase 1 (HPK1)-mediated phosphorylation and ubiquitination of activated B cell linker protein (BLNK).

Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-like serine/threonine kinase that suppresses immune responses and autoimmunity. B cell receptor (BCR) signaling activates HPK1 by inducing BLNK/HPK1 interaction. Whether HPK1 can reciprocally regulate BLNK during BCR signaling is unknown. Here, we show that HPK1-deficient B cells display hyper-proliferation and hyper-activation of IκB kinase and MAPKs (ERK, p38, and JNK) upon the ligation of BCR. HPK1 attenuates BCR-induced cell activation via inducing BLNK threonine 152 phosphorylation, which mediates BLNK/14-3-3 binding. Furthermore, threonine 152-phosphorylated BLNK is ubiquitinated at lysine residues 37, 38, and 42, leading to attenuation of MAPK and IκB kinase activation in B cells during BCR signaling. These results reveal a novel negative feedback regulation of BCR signaling by HPK1-mediated phosphorylation, ubiquitination, and subsequent degradation of the activated BLNK.

High-mobility group A1 proteins inhibit expression of nucleotide excision repair factor xeroderma pigmentosum group A.

Cells that overexpress high-mobility group A1 (HMGA1) proteins exhibit deficient nucleotide excision repair (NER) after exposure to DNA-damaging agents, a condition ameliorated by artificially lowering intracellular levels of these nonhistone proteins. One possible mechanism for this NER inhibition is down-regulation of proteins involved in NER, such as xeroderma pigmentosum complimentation group A (XPA). Microarray and reverse transcription-PCR data indicate a 2.6-fold decrease in intracellular XPA mRNA in transgenic MCF-7 cells overexpressing HMGA1 proteins compared with non-HMGA1-expressing cells. XPA protein levels are also approximately 3-fold lower in HMGA1-expressing MCF-7 cells. Moreover, whereas a >2-fold induction of XPA proteins is observed in normal MCF-7 cells 30 min after UV exposure, no apparent induction of XPA protein is observed in MCF-7 cells expressing HMGA1. Mechanistically, we present both chromatin immunoprecipitation and promoter site-specific mutagenesis evidence linking HMGA1 to repression of XPA transcription via binding to a negative regulatory element in the endogenous XPA gene promoter. Phenotypically, HMGA1-expressing cells exhibit compromised removal of cyclobutane pyrimidine dimer lesions, a characteristic of cells that express low levels of XPA. Importantly, we show that restoring expression of wild-type XPA in HMGA1-expressing cells rescues UV resistance comparable with that of normal MCF-7 cells. Together, these data provide strong experimental evidence that HMGA1 proteins are involved in inhibiting XPA expression, resulting in increased UV sensitivity in cells that overexpress these proteins. Because HMGA1 proteins are overexpressed in most naturally occurring cancers, with increasing cellular concentrations correlating with increasing metastatic potential and poor patient prognosis, the current findings provide new insights into previously unsuspected mechanisms contributing to tumor progression.

High-mobility group A1a protein regulates Ras/ERK signaling in MCF-7 human breast cancer cells.

High-mobility group (HMG) A1 proteins are gene regulatory factors whose overexpression is frequently observed in naturally occurring human cancers. The overexpression of transgenic HMGA1 proteins in cells results in neoplastic transformation and promotes progression to malignant cellular phenotypes. To understand the underlying molecular and biological events involved in these phenomena, we used oligonucleotide microarray analyses to generate an HMGA1a-induced expression profile for approximately 22,000 genes. This gene expression profile was generated using a well-characterized transgenic human MCF-7 mammary adenocarcinoma cell line in which overexpression of transgenic HMGA1 promotes a transition to a more malignant and metastatic phenotype. Microarray expression analyses, together with independent quantitative real-time reverse transcriptase polymerase chain reaction results, indicate that HMGA1a regulates genes involved in the Ras-extracellular signal-related kinase (Ras/ERK) mitogenic signaling pathway, including KIT ligand and caveolins 1 and 2. We also found that many cholesterol biosynthesis genes were decreased in cells overexpressing HMGA1a. Cholesterol depletion, decreased caveolin, and increased KIT ligand expression, are all independently associated with the activation of Ras/ERK signaling. Upon further analysis, we found that sensitivity to epidermal growth factor activation of ERK phosphorylation was significantly higher, and that cholesterol was significantly depleted, in cells overexpressing HMGA1a. The cumulative evidence indicates that one likely mechanism by which the HMGA1a protein promotes malignant changes in cells is through increased sensitivity to the activation of the Ras/ERK signaling pathway.

Human KIT ligand promoter is positively regulated by HMGA1 in breast and ovarian cancer cells.

KIT ligand (KL) and its receptor, c-kit, are coexpressed in many types of cancer cells and have been implicated in tumor growth and angiogenesis. While Sertoli cell-specific regulation of the KL promoter has been well characterized, regulation in cancer cells remains to be elucidated. We recently reported microarray results demonstrating that increased high-mobility group (HMG) A1a protein expression correlates with increased KL transcription in MCF-7 human breast cancer cells. Sequence analysis indicates a potential for multiple HMGA1 binding sites within the human KL promoter. In order to better define the underlying molecular mechanisms that HMGA1 uses to facilitate malignant transformation of cancer cells, we have used a variety of methods to determine whether HMGA1a directly regulates the human KL promoter in breast and ovarian cancer cells. Our results indicate that: (i) KL promoter activity is significantly higher in MCF-7 cells overexpressing HMGA1a; (ii) HMGA1a protein binds to AT-rich regions of the KL promoter DNA both in vitro and in vivo; (iii) mutation of the AT-rich regions inhibits HMGA1a binding in vitro; and (iv) HMGA1a-specific inhibition significantly decreases transcription of KL in OCC1 human ovarian cancer cells. In addition, MCF-7 cells with transgenic HMGA1 overexpression stained positive for the KL protein by immunocytochemistry and immunohistochemistry, and were growth-inhibited by KL neutralization. The cumulative evidence indicates that HMGA1 positively regulates the human KL promoter in breast and ovarian cancer cells and implicates serum KL as a diagnostic marker for HMGA1-positive carcinomas.

Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function.

We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential (Delta Psi(m)). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses.

HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-gamma1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3tau (a phosphorylated serine-binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3tau. Our results indicate that HPK1 negatively regulates TCR signaling and T cell-mediated immune responses.

Dynamic mitochondrial localization of nuclear transcription factor HMGA1.

It has been well established that high mobility group A1 (HMGA1) proteins act within the nucleus of mammalian cells as architectural transcription factors that regulate the expression of numerous genes. Here, however, we report on the unexpected cytoplasmic/mitochondrial localization of the HMGA1 proteins within multiple cell types. Indirect immunofluorescence, electron microscopic immunolocalization, and Western blot studies revealed that, in addition to the nucleus, HMGA1 proteins could also be found in both the cytoplasm and mitochondria of randomly dividing populations of wild-type murine NIH3T3 cells and transgenic human MCF-7 breast cancer epithelial cells expressing a hemagglutinin tagged-HMGA1a fusion protein. While the molecular mechanisms underlying these novel subcellular localization patterns have not yet been determined, initial synchronization studies revealed a dynamic, cell cycle-dependent translocation of HMGA1 proteins from the nucleus into the cytoplasm and mitochondria of NIH3T3 cells. Furthermore, preliminary functionality studies utilizing a modified "chromatin" immunoprecipitation protocol revealed that HMGA1 retains its DNA binding capabilities within the mitochondria and associates with the regulatory D-loop region in vivo. We discuss potential new biological roles for the classically nuclear HMGA1 proteins with regard to the observed nucleocytoplasmic translocation, mitochondrial internalization, and regulatory D-loop DNA binding.

Inhibition of nucleotide excision repair by high mobility group protein HMGA1.

The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-foot-printing" demonstrated that CPD formation within a long run of Ts (T(18)-tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.