The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion.

Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ß1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with α2ß1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with α2ß1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.

MYC activity is negatively regulated by a C-terminal lysine cluster.

The MYC oncogene is not only deregulated in cancer through abnormally high levels of expression, but also through oncogenic lesions in upstream signalling cascades. Modelling MYC deregulation using signalling mutants is a productive research strategy. For example, the MYC threonine-58 to alanine substitution mutant (T58A) within MYC-homology box 1 is more transforming than wild-type (WT) MYC, because of decreased apoptosis and increased protein stability. Understanding the regulatory mechanisms controlling T58 phosphorylation has led to new approaches for the development of MYC inhibitors. In this manuscript, we have extensively characterized a MYC signalling mutant in which six lysine residues near the highly conserved MYC homology box IV and basic region have been substituted to arginines (6KR). Previous literature suggests these lysines can undergo both ubiquitylation and acetylation. We show MYC 6KR is able to fully rescue the slow growth phenotype of HO15.19 MYC-null fibroblasts, and promote cell cycle entry of serum-starved MCF10A cells. Remarkably, 6KR increased anchorage-independent colony growth compared with WT MYC in both SH-EP and MCF10A cells. Moreover, it was also more potent in promoting xenograft tumour growth of Rat1A and SH-EP cells. Combined, our data identify this region and these six lysines as important residues for the negative regulation of MYC-induced transformation. Mechanistically, we demonstrate that, unlike T58A, the increased transformation is not a result of increased protein stability or a reduced capacity for 6KR to induce apoptosis. Through expression analysis and luciferase reporter assays, we show that 6KR has increased transcriptional activity compared with WT MYC. Combined, through a comprehensive evaluation across multiple cell types, we identify an important regulatory region within MYC. A better understanding of the full scope of signalling through these residues will provide further insights into the mechanisms contributing to MYC-induced tumorigenesis and may unveil novel therapeutic strategies to target Myc in cancer.

Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress.

Loss of function of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene is associated with many human cancers. In the cytoplasm, PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K) signaling pathway. PTEN also accumulates in the nucleus, where its function remains poorly understood. We demonstrate that SUMOylation (SUMO, small ubiquitin-like modifier) of PTEN controls its nuclear localization. In cells exposed to genotoxic stress, SUMO-PTEN was rapidly excluded from the nucleus dependent on the protein kinase ataxia telangiectasia mutated (ATM). Cells lacking nuclear PTEN were hypersensitive to DNA damage, whereas PTEN-deficient cells were susceptible to killing by a combination of genotoxic stress and a small-molecule PI3K inhibitor both in vitro and in vivo. Our findings may have implications for individualized therapy for patients with PTEN-deficient tumors.

mTOR signaling to translation.

Over the past few years, the target of rapamycin (TOR) pathway has been implicated in the control of translation, both in yeast and in higher eukaryotes. In this review, we provide an overview of translation in eukaryotes, and discuss the mechanisms and advantages of the regulation of translation. We then describe how the TOR pathway can modulate translation in yeast and in mammals, through the modulation of the phosphorylation of key translation components, and the regulation of the abundance of ribosomes and translation factors.

Hierarchical phosphorylation of the translation inhibitor 4E-BP1.

In most instances, translation is regulated at the initiation phase, when a ribosome is recruited to the 5' end of an mRNA. The eIF4E-binding proteins (4E-BPs) interdict translation initiation by binding to the translation factor eIF4E, and preventing recruitment of the translation machinery to mRNA. The 4E-BPs inhibit translation in a reversible manner. Hypophosphorylated 4E-BPs interact avidly with eIF4E, whereas 4E-BP hyperphosphorylation, elicited by stimulation of cells with hormones, cytokines, or growth factors, results in an abrogation of eIF4E-binding activity. We reported previously that phosphorylation of 4E-BP1 on Thr 37 and Thr 46 is relatively insensitive to serum deprivation and rapamycin treatment, and that phosphorylation of these residues is required for the subsequent phosphorylation of a set of unidentified serum-responsive sites. Here, using mass spectrometry, we identify the serum-responsive, rapamycin-sensitive sites as Ser 65 and Thr 70. Utilizing a novel combination of two-dimensional isoelectric focusing/SDS-PAGE and Western blotting with phosphospecific antibodies, we also establish the order of 4E-BP1 phosphorylation in vivo; phosphorylation of Thr 37/Thr 46 is followed by Thr 70 phosphorylation, and Ser 65 is phosphorylated last. Finally, we show that phosphorylation of Ser 65 and Thr 70 alone is insufficient to block binding to eIF4E, indicating that a combination of phosphorylation events is necessary to dissociate 4E-BP1 from eIF4E.

The major mRNA-associated protein YB-1 is a potent 5' cap-dependent mRNA stabilizer.

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA-associated protein YB-1, also termed p50, is a potent cap-dependent mRNA stabilizer. YB-1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB-1 depletion results in accelerated mRNA decay. The cold shock domain of YB-1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5' end is required for YB-1-mediated stabilization. Significantly, exogenously added YB-1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB-1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down-regulation of eIF4E activity or increasing the YB-1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.

The target of rapamycin (TOR) proteins.

Rapamycin potently inhibits downstream signaling from the target of rapamycin (TOR) proteins. These evolutionarily conserved protein kinases coordinate the balance between protein synthesis and protein degradation in response to nutrient quality and quantity. The TOR proteins regulate (i) the initiation and elongation phases of translation, (ii) ribosome biosynthesis, (iii) amino acid import, (iv) the transcription of numerous enzymes involved in multiple metabolic pathways, and (v) autophagy. Intriguingly, recent studies have also suggested that TOR signaling plays a critical role in brain development, learning, and memory formation.

Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.

The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).

Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.

eIF4E activity is regulated at multiple levels.

A key regulatory step in translation is initiation, or the recruitment of the translational machinery to the 5' end of mRNA. The 5' terminus of most mRNAs is demarcated by a m7GpppN cap (where m is a methyl group, and N is any nucleotide). The m7 cap is essential for the translation of most mRNAs, as it directs the translational machinery to the 5' end of the mRNA via its interaction with the cap binding protein, the eukaryotic translation initiation factor 4E (eIF4E). eIF4E is the limiting initiation factor in most cells. Thus, eIF4E activity plays a principal role in determining global translation rates. Consistent with this role, eIF4E is required for cell cycle progression, exhibits anti-apoptotic activity, and, when overexpressed, transforms cells. This review focuses upon the various mechanisms utilized in the regulation of eIF4E activity. (1) eIF4E is regulated transcriptionally; it is one of the few identified transcriptional targets of c-myc. (2) eIF4E is phosphorylated following activation of the MNK1 kinase, a substrate of the ERK and p38 MAPKs. The recent determination of the three-dimensional structure of eIF4E bound to a m7 cap analog has provided insight into the mechanisms involved in the regulation of the eIF4E-cap and eIF4E-mRNA interactions. As suggested by the crystal structure, phosphorylation of eIF4E may enhance its affinity for mRNA. (3) eIF4E is also regulated through binding to a family of translational repressor proteins. Interaction with the 4E-BPs prevents the incorporation of eIF4E into an active translation initiation complex, and thus, inhibits cap-dependent translation. This inhibitory interaction is relieved following phosphorylation of the 4E-BPs by a PI3K-dependent pathway, involving signalling by the anti-apoptotic kinase Akt/PKB, as well as FRAP/mTOR.

eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation.

Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex that mediates recruitment of ribosomes to mRNA. This event is the rate-limiting step for translation under most circumstances and a primary target for translational control. Functions of the constituent proteins of eIF4F include recognition of the mRNA 5' cap structure (eIF4E), delivery of an RNA helicase to the 5' region (eIF4A), bridging of the mRNA and the ribosome (eIF4G), and circularization of the mRNA via interaction with poly(A)-binding protein (eIF4G). eIF4 activity is regulated by transcription, phosphorylation, inhibitory proteins, and proteolytic cleavage. Extracellular stimuli evoke changes in phosphorylation that influence eIF4F activity, especially through the phosphoinositide 3-kinase (PI3K) and Ras signaling pathways. Viral infection and cellular stresses also affect eIF4F function. The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiated and regulated. Evidence suggests that eIF4F is also implicated in malignancy and apoptosis.

C/EBPbeta, but not C/EBPalpha, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland.

The CCAAT/enhancer binding proteins (C/EBPs) are differentially expressed throughout mammary gland development and interact with binding sites within the promoter of a milk protein gene, beta-casein. The specific roles of C/EBPbeta and C/EBPalpha in mouse mammary gland development and differentiation have been investigated in mice that carry targeted deletions of these genes. C/EBPbeta-/- virgin mice exhibited cystic, enlarged mammary ducts with decreased secondary branching. Transplantation of C/EBPbeta-/- mammary epithelium into the cleared mammary fat pads of nude mice confirmed that this defect in ductal morphogenesis was intrinsic to the epithelium. When treated with estrogen/progesterone (E+P) to simulate pregnancy, C/EBPbeta-/- mammary glands displayed only limited lobuloalveolar development and ductal side branching. Primary mammary epithelial cells obtained from E+P-treated C/EBPbeta-/- mice that were cultured on extracellular matrix gels did not functionally differentiate in response to lactogenic hormones despite their organization into three-dimensional structures. Expression of beta-casein protein was inhibited 85%-100% and whey acidic protein (WAP) was undetectable. In contrast, no detectable alterations in mammary development or beta-casein expression were observed in mammary outgrowths derived from newborn C/EBPalpha-/- mammary epithelium transplanted into the cleared mammary fat pads of syngeneic hosts. These results demonstrate that C/EBPbeta, but not C/EBPalpha, is required for ductal morphogenesis, lobuloalveolar development, and functional differentiation of mammary epithelial cells.

Composite response elements mediate hormonal and developmental regulation of milk protein gene expression.

Our laboratory has been studying the mechanisms by which hormones regulate the expression of differentiated function in the normal mammary gland and how these regulatory mechanisms have deviated in breast cancer. Two rat milk protein genes, encoding beta-casein and whey acidic protein, have been employed as molecular markers of mammary epithelial cell differentiation. Composite response elements containing multiple binding sites for several transcription factors mediate the hormonal and developmental regulation of milk protein gene expression. In the whey protein gene promoters, these include binding sites for nuclear factor (NF)-I, as well as the glucocorticoid receptor (GR) and signal transducers and activators of transcription (Stat5). In the casein promoters, these include binding sites for Stat5, Yin Yang 1 (YY1), GR and the CCAAT/enhancer binding protein (C/EBP). The C/EBP family of DNA binding proteins may play a pivotal role in maintaining the balance between cell proliferation and terminal differentiation in mammary epithelial cells. During normal mammary gland development, expression of LIP (liver-enriched inhibitory protein, a dominant-negative isoform of C/EBP beta) is hormonally regulated and correlates with cell proliferation during pregnancy. LIP can form heterodimers with other C/EBP family members and suppress their transcriptional activity. In contrast, C/EBP alpha is predominantly expressed during lactation following terminal differentiation. Elevated LIP levels have been detected in mouse, rat and human breast tumours of different aetiologies. This provides a mechanism, therefore, to block terminal differentiation and facilitate continued proliferation.

A novel functional human eukaryotic translation initiation factor 4G.

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.

Expression of a translationally regulated, dominant-negative CCAAT/enhancer-binding protein beta isoform and up-regulation of the eukaryotic translation initiation factor 2alpha are correlated with neoplastic transformation of mammary epithelial cells.

A translationally regulated, dominant-negative isoform of CCAAT/enhancer-binding protein beta is expressed in transplantable and primary mouse mammary tumors of different etiologies but is not expressed in preneoplastic mammary hyperplasias or in primary prostate, lung, lens, ovary or lymphoid tumors. The eukaryotic initiation factor 2alpha protein is also expressed at significantly higher levels (69.8 +/- 7.2%) in these mammary tumors compared with normal and hyperplastic tissues. Thus, misregulation of eukaryotic initiation factor 2alpha may promote the expression of a dominant-negative CCAAT/enhancer-binding protein beta isoform, which may inhibit terminal differentiation and facilitate uncontrolled proliferation of mammary epithelial cells.

The mammary gland as a bioreactor: factors regulating the efficient expression of milk protein-based transgenes.

Specific regulatory regions required for hormonal regulation and tissue-specific expression of rat beta-casein and why acidic protein (WAP) genes in the mammary gland have been defined. Composite response elements with multiple binding sites for several transcription factors have been identified. Mammary gland-specific gene expression appears not to be mediated by a single transcription factor, but instead requires cooperative interactions among several factors. Signal transduction pathways regulated by lactogenic hormones result in transcription factor binding and interaction within these elements, chromatin-structure changes, and milk-protein gene expression. Intragenic sequences in the 5' and 3' untranslated regions of the beta-casein and WAP mRNAs, respectively, also appear crucial for the efficient expression of these genes. Vectors to target the expression of heterologous genes, such as insulin-like growth factor I, to the mammary gland can be designed. This technology can be used to manipulate milk composition in transgenic animals, one result being improved infant formulas.

Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element.

alpha 2-Macroglobulin (alpha 2M) is expressed at high levels in the corpus luteum of pregnant rats in response to PRL and rat placental lactogens. These studies document that PRL induction of alpha 2M mRNA occurs rapidly in granulosa cells differentiated to the preovulatory phenotype in the presence of FSH and steroid, is hormone specific [induced by PRL but not by LH or interleukin-6 (IL-6)], and involves tyrosine kinase activity. To analyze the cellular signaling events stimulated by PRL, transient transfections of granulosa cells and electrophoretic mobility shift assays were done using the IL-6 response element (IL-6RE) of the alpha 2M promoter. The IL-6RE consists of two gamma-activating like sequences (GAS) that bind the acute phase response factor (APRF/Stat 3) in rat liver and the mammary gland factor (MGF/Stat 5) from mammary tissue. By transfecting various alpha 2M promoter-luciferase reporter transgenes into the granulosa cell cultures, we show that the GAS-like sites together with the minimal -48 base pairs of the alpha 2M promoter can confer PRL inducibility to the luciferase reporter gene. These same GAS-like sequences of the alpha 2M promoter were used to analyze the DNA-binding activity of proteins in whole cell extracts prepared from differentiated granulosa cells exposed to PRL for 0.25, 0.5, 4, and 20 h. PRL rapidly stimulated the binding of a specific protein to labeled alpha 2M GAS-like oligonucleotide, and this PRL-induced binding activity was shown to contain Stat 5 but not Stat 1 or Stat 3, using specific antibodies in the electrophoretic mobility shift assays. Because both Stat 5 and Stat 3 proteins are present in the whole cell extracts of differentiated granulosa cells, PRL appears to activate detectable amounts of Stat 5 (and not Stat 3). Thus, the initial induction of the alpha 2M gene by PRL in differentiated rat granulosa cells involves, at least in part, the activation (tyrosine phosphorylation?) of Stat 5.

Regulation of mammary gland factor/Stat5a during mammary gland development.

The rat homolog of sheep mammary gland factor (MGF)/Stat5 has been isolated and used to study the regulation of Stat5 during mammary gland development and PRL regulation in COS cells transfected with Stat5a and the PRL receptor. Two alternatively spliced isoforms, designated Stat5a1 and Stat5a2, were identified, the latter encoding a carboxy-terminal truncated protein. A polyclonal antibody to a carboxy-terminal peptide of Stat5a1 was generated and used to measure the level of this isoform during mammary gland development and after PRL induction in COS cells transiently transfected with Stat5a and the long form of the PRL receptor. Surprisingly, Stat5a mRNA and protein were readily detected both in virgin rats and after mammary gland involution. The levels of Stat5a increased during pregnancy, were highest in late pregnancy, and then, unexpectedly, decreased during lactation, the time at which the highest levels of milk protein gene expression are observed. Electrophoretic mobility shift assays using the specific anti-Stat5a1 antisera demonstrated that Stat5a1 comprises part of the heterogeneous, PRL-inducible, protein-DNA complex associated with the beta-casein GAS site. Immunocytochemical analysis detected considerable cytoplasmic and some nuclear staining for Stat5a1 during late pregnancy and predominantly nuclear staining during early lactation. The lack of correspondence of Stat5a gene expression and beta-casein gene expression suggests that Stat5 activation may facilitate the interaction of other factors binding within composite response elements identified recently in the milk protein gene promoters that are then responsible for the stable expression of milk protein genes in terminally differentiated mammary epithelial cells.