A Unique Family of Neuronal Signaling Proteins Implicated in Oncogenesis and Tumor Suppression.
ABSTRACT: The neuronal proteins GAP43 (neuromodulin), MARCKS, and BASP1 are highly expressed in the growth cones of nerve cells where they are involved in signal transmission and cytoskeleton organization. Although their primary structures are unrelated, these signaling proteins share several structural properties like fatty acid modification, and the presence of cationic effector domains. GAP43, MARCKS, and BASP1 bind to cell membrane phospholipids, a process reversibly regulated by protein kinase C-phosphorylation or by binding to the calcium sensor calmodulin (CaM). GAP43, MARCKS, and BASP1 are also expressed in non-neuronal cells, where they may have important functions to manage cytoskeleton architecture, and in case of MARCKS and BASP1 to act as cofactors in transcriptional regulation. During neoplastic cell transformation, the proteins reveal differential expression in normal vs. tumor cells, and display intrinsic tumor promoting or tumor suppressive activities. Whereas GAP43 and MARCKS are oncogenic, tumor suppressive functions have been ascribed to BASP1 and in part to MARCKS depending on the cell type. Like MARCKS, the myristoylated BASP1 protein is localized both in the cytoplasm and in the cell nucleus. Nuclear BASP1 participates in gene regulation converting the Wilms tumor transcription factor WT1 from an oncoprotein into a tumor suppressor. The BASP1 gene is downregulated in many human tumor cell lines particularly in those derived from leukemias, which display elevated levels of WT1 and of the major cancer driver MYC. BASP1 specifically inhibits MYC-induced cell transformation in cultured cells. The tumor suppressive functions of BASP1 and MARCKS could be exploited to expand the spectrum of future innovative therapeutic approaches to inhibit growth and viability of susceptible human tumors.
Project description:Cell transformation by the Myc oncoprotein involves transcriptional activation or suppression of specific target genes with intrinsic oncogenic or tumor-suppressive potential, respectively. We have identified the BASP1 (CAP-23, NAP-22) gene as a novel target suppressed by Myc. The acidic 25-kDa BASP1 protein was originally isolated as a cortical cytoskeleton-associated protein from rat and chicken brain, but has also been found in other tissues and subcellular locations. BASP1 mRNA and protein expression is specifically suppressed in fibroblasts transformed by the v-myc oncogene, but not in cells transformed by other oncogenic agents. The BASP1 gene encompasses 2 exons separated by a 58-kbp intron and a Myc-responsive regulatory region at the 5' boundary of untranslated exon 1. Bicistronic expression of BASP1 and v-myc from a retroviral vector blocks v-myc-induced cell transformation. Furthermore, ectopic expression of BASP1 renders fibroblasts resistant to subsequent cell transformation by v-myc, and exogenous delivery of the BASP1 gene into v-myc-transformed cells leads to significant attenuation of the transformed phenotype. The inhibition of v-myc-induced cell transformation by BASP1 also prevents the transcriptional activation or repression of known Myc target genes. Mutational analysis showed that the basic N-terminal domain containing a myristoylation site, a calmodulin binding domain, and a putative nuclear localization signal is essential for the inhibitory function of BASP1. Our results suggest that down-regulation of the BASP1 gene is a necessary event in myc-induced oncogenesis and define the BASP1 protein as a potential tumor suppressor.
Project description:The MYC protein is a transcription factor with oncogenic potential controlling fundamental cellular processes such as cell proliferation, metabolism, differentiation, and apoptosis. The MYC gene is a major cancer driver, and elevated MYC protein levels are a hallmark of most human cancers. We have previously shown that the brain acid-soluble protein 1 gene (BASP1) is specifically downregulated by the v-myc oncogene and that ectopic BASP1 expression inhibits v-myc-induced cell transformation. The 11-amino acid effector domain of the BASP1 protein interacts with the calcium sensor calmodulin (CaM) and is mainly responsible for this inhibitory function. We also reported recently that CaM interacts with all MYC variant proteins and that ectopic CaM increases the transactivation and transformation potential of the v-Myc protein. Here, we show that the presence of excess BASP1 or of a synthetic BASP1 effector domain peptide leads to displacement of v-Myc from CaM. The protein stability of v-Myc is decreased in cells co-expressing v-Myc and BASP1, which may account for the inhibition of v-Myc. Furthermore, suppression of v-Myc-triggered transcriptional activation and cell transformation is compensated by ectopic CaM, suggesting that BASP1-mediated withdrawal of CaM from v-Myc is a crucial event in the inhibition. In view of the tumor-suppressive role of BASP1 which was recently also reported for human cancer, small compounds or peptides based on the BASP1 effector domain could be used in drug development strategies aimed at tumors with high MYC expression.
Project description:The Wilms' tumor-1 protein (WT1) is a transcriptional regulator that can either activate or repress genes controlling cell growth, apoptosis and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and mediates WT1's transcriptional repression activity. BASP1 is contained within large complexes, suggesting that it works in concert with other factors. Here we report that the transcriptional repressor prohibitin is part of the WT1-BASP1 transcriptional repression complex. Prohibitin interacts with BASP1, colocalizes with BASP1 in the nucleus, and is recruited to the promoter region of WT1 target genes to elicit BASP1-dependent transcriptional repression. We demonstrate that prohibitin and BASP1 cooperate to recruit the chromatin remodeling factor BRG1 to WT1-responsive promoters and that this results in the dissociation of CBP from the promoter region of WT1 target genes. As seen with BASP1, prohibitin can associate with phospholipids. We demonstrate that the recruitment of PIP2 and HDAC1 to WT1 target genes is also dependent on the concerted activity of BASP1 and prohibitin. Our findings provide new insights into the function of prohibitin in transcriptional regulation and uncover a BASP1-prohibitin complex that plays an essential role in the PIP2-dependent recruitment of chromatin remodeling activities to the promoter.
Project description:The Wilms' tumor 1 protein WT1 is a transcriptional regulator that is involved in cell growth and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and converts WT1 from a transcriptional activator to a repressor. Here, we demonstrate that the N-terminal myristoylation of BASP1 is required in order to elicit transcriptional repression at WT1 target genes. We show that myristoylated BASP1 binds to nuclear PIP2, which leads to the recruitment of PIP2 to the promoter regions of WT1-dependent target genes. BASP1's myristoylation and association with PIP2 are required for the interaction of BASP1 with HDAC1, which mediates the recruitment of HDAC1 to the promoter and elicits transcriptional repression. Our findings uncover a role for myristoylation in transcription, as well as a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase.
Project description:WT1 is a transcriptional activator that controls the boundary between multipotency and differentiation. The transcriptional cofactor BASP1 binds to WT1, forming a transcriptional repressor complex that drives differentiation in cultured cells; however, this proposed mechanism has not been demonstrated in vivo. We used the peripheral taste system as a model to determine how BASP1 regulates the function of WT1. During development, WT1 is highly expressed in the developing taste cells while BASP1 is absent. By the end of development, BASP1 and WT1 are co-expressed in taste cells, where they both occupy the promoter of WT1 target genes. Using a conditional BASP1 mouse, we demonstrate that BASP1 is critical to maintain the differentiated state of adult taste cells and that loss of BASP1 expression significantly alters the composition and function of these cells. This includes the de-repression of WT1-dependent target genes from the Wnt and Shh pathways that are normally only transcriptionally activated by WT1 in the undifferentiated taste cells. Our results uncover a central role for the WT1-BASP1 complex in maintaining cell differentiation in vivo.
Project description:The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.
Project description:We have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our analysis reveals that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We found that WT1 and BASP1 can divert the differentiation program of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. Cell lines were generated expressing the BASP1 gene from the vector pcDNA3. Array analysis was performed on 4 conditions: cells expressing BASP1 and controls, in the absence and presence of Phorbol 12-myristate 13-acetate (PMA).
Project description:BACKGROUND:Pancreatic cancer is a heterogenous disease with a poor prognosis. This study aimed to discover and validate prognostic tissue biomarkers in pancreatic cancer using a mass spectrometry (MS) based proteomics approach. METHODS:Global protein sequencing of fresh frozen pancreatic cancer and healthy pancreas tissue samples was conducted by MS to discover potential protein biomarkers. Selected candidate proteins were further verified by targeted proteomics using parallel reaction monitoring (PRM). The expression of biomarker candidates was validated by immunohistochemistry in a large tissue microarray (TMA) cohort of 141 patients with resectable pancreatic cancer. Kaplan-Meier and Cox proportional hazard modelling was used to investigate the prognostic utility of candidate protein markers. FINDINGS:In the initial MS-discovery phase, 165 proteins were identified as potential biomarkers. In the subsequent MS-verification phase, a panel of 45 candidate proteins was verified by the development of a PRM assay. Brain acid soluble protein 1 (BASP1) was identified as a new biomarker candidate for pancreatic cancer possessing largely unknown biological and clinical functions and was selected for further analysis. Importantly, bioinformatic analysis indicated that BASP1 interacts with Wilms tumour protein (WT1) in pancreatic cancer. TMA-based immunohistochemistry analysis showed that BASP1 was an independent predictor of prolonged survival (HR 0.468, 95% CI 0.257-0.852, p = .013) and predicted favourable response to adjuvant chemotherapy, whereas WT1 indicated a worsened survival (HR 1.636, 95% CI 1.083-2.473, p = .019) and resistance to chemotherapy. Interaction analysis showed that patients with negative BASP1 and high WT1 expression had the poorest outcome (HR 3.536, 95% CI 1.336-9.362, p = .011). INTERPRETATION:We here describe an MS-based proteomics platform for developing biomarkers for pancreatic cancer. Bioinformatic analysis and clinical data from our study suggest that BASP1 and its putative interaction partner WT1 can be used as biomarkers for predicting outcomes in pancreatic cancer patients.
Project description:The cysteine protease caspase-3, best known as an executioner of cell death in apoptosis, also plays a non-apoptotic role in N-methyl-d-aspartate receptor-dependent long-term depression of synaptic transmission (NMDAR-LTD) and ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor endocytosis in neurons. The mechanism by which caspase-3 regulates LTD and AMPA receptor endocytosis, however, remains unclear. Here, we addressed this question by using an enzymatic N-terminal peptide enrichment method and mass spectrometry to identify caspase-3 substrates in neurons. Of the many candidates revealed by this proteomic study, we have confirmed BASP1, Dbn1, and Gap43 as true caspase-3 substrates. Moreover, in hippocampal neurons, Gap43 mutants deficient in caspase-3 cleavage inhibit AMPA receptor endocytosis and LTD. We further demonstrated that Gap43, a protein well-known for its functions in axons, is also localized at postsynaptic sites. Our study has identified Gap43 as a key caspase-3 substrate involved in LTD and AMPA receptor endocytosis, uncovered a novel postsynaptic function for Gap43 and provided new insights into how long-term synaptic depression is induced.
Project description:The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.