Oestrogen receptor (ER) is a good prognostic marker for the treatment of breast cancers. Upregulation of metastatic tumour antigen 1 (MTA1) is associated with the invasiveness and metastatic potential of several human cancers and acts as a co-repressor of nuclear ER-alpha. Here we identify a naturally occurring short form of MTA1 (MTA1s) that contains a previously unknown sequence of 33 amino acids with an ER-binding motif, Leu-Arg-Ile-Leu-Leu (LRILL). MTA1s localizes in the cytoplasm, sequesters ER in the cytoplasm, and enhances non-genomic responses of ER. Deleting the LRILL motif in MTA1s abolishes its co-repressor function and its interaction with ER, and restores nuclear localization of ER. Dysregulation of human epidermal growth factor receptor-2 in breast cancer cells enhances the expression of MTA1s and the cytoplasmic sequestration of ER. Expression of MTA1s in breast cancer cells prevents ligand-induced nuclear translocation of ER and stimulates malignant phenotypes. MTA1s expression is increased in human breast tumours with no or low nuclear ER. The regulation of the cellular localization of ER by MTA1s represents a mechanism for redirecting nuclear receptor signalling by nuclear exclusion.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAF(V600E) has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAF(V600E) also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAP kinase inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAP kinase pathway-driven re-localization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells, and provides further evidence to support testing therapeutic strategies combining MAP kinase pathway inhibition with immunotherapies in the clinical setting.

Here, we investigated the role of P21-activated kinase 1 (Pak1) signaling in the function of estrogen receptor-A (ER-A) as assessed by serine 305 (S305) activation and transactivation activity of ER. We found that Pak1 overexpression interfered with the antiestrogenic action of tamoxifen upon the ER transactivation function in hormone-sensitive cells. In addition, tamoxifen stimulation led to up-regulation of ER target genes in breast cancer cells with increased Pak1 expression. Tamoxifen also increased Pak1-ER interaction in tamoxifenresistant but not in tamoxifen-sensitive cells. Results from the mutational studies discovered a role of ER-S305 phosphorylation in triggering a subsequent phosphorylation of serine 118 (S118), and these effects were further potentiated by tamoxifen treatment. We found that S305 activation-linked ER transactivation function requires a functional S118, and active Pak1 signaling is required for a sustaining S118 phosphorylation of the endogenous ER. All of these events were positively influenced by tamoxifen and thus may contribute toward the loss of antiestrogenic effect of tamoxifen. These findings suggest that Pak1 signaling-dependent activation of ER-S305 leads to an enhanced S118 phosphorylation presumably due to a conformational change, and such structural modifications may participate in the development of tamoxifen resistance.

Transcription, splicing, and translation are potentially coordinately regulatable in a temporospatial-dependent manner, although supporting experimental evidence for this notion is scarce. Yeast two-hybrid screening of a mammary gland cDNA library with human p21-activated kinase 1 (Pak1) as bait identified polyC-RNAbinding protein 1 (PCBP1), which controls translation from mRNAs containing the DICE (differentiation control element). Mitogenic stimulation of human cells phosphorylated PCBP1 on threonines 60 and 127 in a Pak1-sensitive manner. Pak1-dependent phosphorylation of PCBP1 released its binding and translational inhibition from a DICE-minigene. Overexpression of PCBP1 also inhibited the translation of the endogenous L1 cell adhesion molecule mRNA, which contains two DICE motifs in the 3 untranslated region. We also found that Pak1 activation led to an increased nuclear retention of PCBP1, recruitment to the eukaryotic translation initiation factor 4E (eIF4E) promoter, and stimulation of eIF4E expression in a Pak1-sensitive manner. Moreover, mitogenic stimulation promoted Pak1-and PCBP1-dependent alternative splicing and exon inclusion from a CD44 minigene. The alternative splicing functions of PCBP1 were in turn mediated by its intrinsic interaction with Caper ␣, a U2 snRNP auxiliary factor-related protein previously implicated in RNA splicing. These findings establish the principle that a single coregulator can function as a signal-dependent and coordinated regulator of transcription, splicing, and translation. differentiation control element ͉ eukaryotic translation initiation factor 4E ͉ p21-activated kinase 1

Here we define a function of metastasis-associated protein 1 (MTA1), a presumed corepressor of estrogen receptor α (ERα), as a transcriptional activator of Breast Cancer Amplified Sequence 3 (BCAS3), a gene amplified and overexpressed in breast cancers. We identified BCAS3 as a MTA1 chromatin target in a functional genomic screen. MTA1 stimulation of BCAS3 transcription required ERα and involved a functional ERE half-site in BCAS3 . Furthermore, we discovered that MTA1 is acetylated on lysine 626, and that this acetylation is necessary for a productive transcriptional recruitment of RNA polymerase II complex to the BCAS3 enhancer sequence. BCAS3 expression was elevated in mammary tumors from MTA1 transgenic mice and 60% of the human breast tumors, and correlated with the coexpression of MTA1 as well as with tumor grade and proliferation of primary breast tumor samples. These findings reveal a previously unrecognized function of MTA1 in stimulating BCAS3 expression and suggest an important role for MTA1-BCAS3 pathway in promoting cancerous phenotypes in breast tumor cells.

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.

Here, we provide gain-of-function, loss-of function, and molecular evidence supporting genetic interactions between metastasis associated protein 1 (MTA1) and Six3 and between Six3 and rhodopsin. We discovered that MTA1 physically interacts with the Six3 chromatin in a histone deacetylase-dependent manner, leading to transcriptional suppression of the Six3 gene. MTA1 is also a Six3-interacting corepressor that contributes to a self-negative regulation of Six3 transcription by Six3. In contrast, deletion of the MTA1 alleles in murine embryonic fibroblasts or its knockdown in rat retinal ganglion cells stimulates Six3 expression. MTA1 inactivation in the MTA1-null mice results in an elevated Six3 level and proliferation of the retina cells with no obvious abnormities in eye formation. However, unexpectedly, we discovered an enhanced recruitment of Six3 to the rhodopsin chromatin in retina from the MTA1-null mice; Six3's homeodomain interacts with specific DNA elements in the rhodopsin promoter to stimulate its transcription, resulting in increased rhodopsin expression. Further, in holoprosencephaly patients, Six3 protein with a naturally occurring deletion mutation in the helix 3 of the homeodomain does not bind to rhodopsin DNA or stimulate rhodopsin transcription, implying a potential defective rhodopsin pathway in the affected holoprosencephaly patients. Further Six3 cooperates with Crx or NRL in stimulating transcription from the rhodopsin-luc. These findings reveal a previously unrecognized role for the MTA1 as an upstream modifier of Six3 and indicate that Six3 is a direct stimulator of rhodopsin expression, thus revealing a putative role for the MTA1/ Six3/rhodopsin pathway in vertebrate eye.MTA1 ͉ transcription repressor ͉ histone deacetylase

Integrin-linked kinase (ILK) is a phosphorylated protein that regulates physiological processes that overlap with those regulated by p21-activated kinase 1 (PAK1). Here we report the possible role of ILK phosphorylation by PAK1 in ILK-mediated signaling and intracellular translocation. We found that PAK1 phosphorylates ILK at threonine-173 and serine-246 in vitro and in vivo. Depletion of PAK1 decreased the levels of endogenous ILK phosphorylation in vivo. Mutation of PAK1 phosphorylation sites on ILK to alanine reduced cell motility and cell proliferation. Biochemical fractionation, confocal microscopy, and chromatin-interaction analyses of human cells revealed that ILK localizes predominantly in the cytoplasm but also resides in the nucleus. Transfection of MCF-7 cells with point mutants ILK-T173A, ILK-S246A, or ILK-T173A; S246A (ILK-DM) altered ILK localization. Selective depletion of PAK1 dramatically increased the nuclear and focal point accumulation of ILK, further demonstrating a role for PAK1 in ILK translocation. We also identified functional nuclear localization sequence and nuclear export sequence motifs in ILK, delineated an apparently integral role for ILK in maintaining normal nuclear integrity, and established that ILK interacts with the regulatory region of the CNKSR3 gene chromatin to negatively modulate its expression. Together, these results suggest that ILK is a PAK1 substrate, undergoes phosphorylation-dependent shuttling between the cell nucleus and cytoplasm, and interacts with gene-regulatory chromatin.p21-activated kinase 1 ͉ signaling ͉ nucleus T he integrin-linked kinase (ILK) is a major signaling integrator in mammalian cells. It plays critical roles in cell motility, adhesion-dependent signaling, cytoskeleton reorganization, and tumor progression and invasion (1-3). Upon activation by different stimuli, ILK phosphorylates AKT and glycogen synthase kinase 3␤ and participates in the related signaling cascades (1). ILK localizes to the focal adhesions by binding to the cytoplasmic tail of the ␤1-integrin subunit and by association with several cytoskeleton proteins, creating an ILK-based scaffold complex important for linking extracellular stimuli with cytoskeleton remodeling (1, 4). In addition to the focal adhesion points, ILK also localizes in the cytoplasm (5) and even in the nuclear compartment upon deletion of the caveolin-1-interacting site in ILK (6). Mutational analyses of the ILK kinase domain suggest that the scaffolding and enzymatic properties of ILK are intimately linked (4) and consequently that ILK's subcellular localization might depend on posttranslational modification of specific ILK residues. Nevertheless, even though ILK autophosphorylation of serine-343 within the kinase domain is considered important for ILK functions (7), regulation of ILK by direct phosphorylation has not yet been demonstrated. Moreover, even though ILK is known to be a phosphorylated protein, neither its upstream kinase, the biochemical basis for its spatiotemporal localization, nor the regulat...