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Daniela Gardiol - One of the best experts on this subject based on the ideXlab platform.
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differential expression of DLG1 as a common trait in different human diseases an encouraging issue in molecular pathology
Biological Chemistry, 2019Co-Authors: Federico Marziali, Maria Paula Dizanzo, Ana Laura Cavatorta, Daniela GardiolAbstract:Human disc large (DLG1) is a scaffolding protein that through the interaction with diverse cell partners participates in the control of key cellular processes such as polarity, proliferation and migration. Experimental data have mainly identified DLG1 as a tumor suppressor. An outstanding point for DLG1 protein is that altered DLG1 expression and DLG1 gene mutations were observed in different pathologies, including cancer and neurological and immunological disorders. Evident changes in DLG1 abundance and/or cell localization were identified in a number of studies suggesting its participation in molecular mechanisms responsible for the development of such illnesses. In this review, we focus on some of the latest findings regarding DLG1 alterations in different diseases as well as its potential use as a biomarker for pathological progression. We further address the current knowledge on the molecular mechanisms regulating DLG1 expression and the posttranslational modifications that may affect DLG1 cell localization and functions. Despite the advances in this field, there are still open questions about the precise molecular link between alterations in DLG1 expression and the development of each specific pathology. The complete understanding of this concern will give us new scenarios for the design of promising diagnosis and therapeutic tools.
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DLG1 polarity protein expression associates with the disease progress of low grade cervical intraepithelial lesions
Experimental and Molecular Pathology, 2017Co-Authors: Ana Laura Cavatorta, Federico Marziali, Alejandra Di Gregorio, Marina Paula Bugnon Valdano, Mariela Cabral, Hebe Bottai, Jorge Cittadini, Ana Lia Nocito, Daniela GardiolAbstract:Human Disc large tumor suppressor (DLG1) participates in regulating cell polarity and proliferation, suggesting an important connection between epithelial organization and cellular growth control. However, it was demonstrated that DLG1 could acquire oncogenic attributes in some specific contexts. In this work, we evaluated the expression of DLG1 and its contribution to the progress of cervical lesions in order to investigate a potential role of this polarity protein in human oncogenic processes. We analyzed cervical biopsies from women with low-grade squamous intraepithelial lesion (LSIL) diagnosis (n=30), for DLG1 expression by immunohistochemistry. These results were correlated with the clinical monitoring of the patients during a 24-month follow-up period. Our data indicate that while all LSIL patients with a DLG1 staining pattern similar to normal tissues are significantly more likely to regress (n=23, Pattern I), all LSIL biopsy specimens showing a diffuse and intense DLG1 staining likely progress to high-grade lesions (n=4, Pattern II). Finally, all persistent LSIL analyzed showed an undetermined DLG1 staining, with a diffuse distribution without a strong intensity (n=3, Pattern III). We found a significant association between the expression pattern of DLG1 and the evolution of the lesion (p<0.00001). This work contributes to the knowledge of DLG1 biological functions, suggesting that its expression may have an important role in the progression of early dysplastic cervical lesions, giving prognostic information.
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regulation of translational efficiency by different splice variants of the disc large 1 oncosuppressor 5 utr
FEBS Journal, 2011Co-Authors: Ana Laura Cavatorta, Federico Marziali, Marina Paula Bugnon Valdano, Lawrence Banks, Florencia Natalia Facciuto, Adriana A Giri, Daniela GardiolAbstract:Human Disc large (DLG1) has been demonstrated to be involved in the control of cell polarity and maintenance of tissue architecture, and is frequently lost in human tumours. However, the mechanisms controlling DLG1 expression are poorly understood. To further examine the regulation of DLG1 expression, we analysed the 5' ends of DLG1 transcripts by rapid amplification of cDNA ends polymerase chain reaction. We identified an alternative splicing event in the 5' region of DLG1 mRNA that generates transcripts with two different 5' untranslated regions (5'-UTRs). We show by reporter assays that the DLG1 5'-UTR containing an alternatively spliced exon interferes with the translation of a downstream open reading frame (ORF). However, no significant differences in mRNA stability among the DLG1 5'-UTR variants were observed. Sequence analysis of the additional exon present in the larger DLG1 5'-UTR showed the presence of an upstream short ORF which is lost in the short version of the 5'-UTR DLG1. By mutagenesis and luciferase assays, we analysed the contribution of this upstream short ORF in reducing translation efficiency, and showed that its disruption can revert, to some extent, the negative regulation of large 5'-UTR. Using computational modelling we also show that the large DLG1 5'-UTR isoform forms a more stable structure than the short version, and this may contribute to its ability to repress translation. This represents the first analysis of the 5' region of the DLG1 transcripts and shows that differential expression of alternatively spliced 5'-UTRs with different translational properties could result in changes in DLG1 abundance.
Xiaolan Qian - One of the best experts on this subject based on the ideXlab platform.
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Abstract 1762: The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1
Molecular and Cellular Biology Genetics, 2019Co-Authors: Beatriz Sanchez-solana, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Parthibane Velayoudame, Jairaj Acharya, Douglas R LowyAbstract:The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain. We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1. We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1. Citation Format: Beatriz Sanchez-Solana, Dunrui Wang, Xiaolan Qian, Alex Papageorge, Parthibane Velayoudame, Jairaj Acharya, Douglas R. Lowy. The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1762.
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Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1.
The Journal of cell biology, 2017Co-Authors: Brajendra K Tripathi, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Ming Zhou, Tiera Grant, Philipp Mertins, Sergey G. Tarasov, Kent W. Hunter, Steven A. CarrAbstract:We report several receptor tyrosine kinase (RTK) ligands increase RhoA-guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.
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Abstract 2155: Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma
Cancer Research, 2015Co-Authors: Brajendra K Tripathi, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Tiera Grant, Philipp Mertins, Steven A. Carr, Douglas R LowyAbstract:The oncogenes SRC and ERK1 are frequently activated in a wide variety of human cancer, while the tumor suppressor DLC1, which encodes a Rho-GAP (GTPase activating protein) essential for its tumor suppressor functions, is frequently down-regulated. However, no prior research has mechanistically linked SRC and ERK1 to DLC1. In this study, we report that SRC and ERK1 cooperate to attenuate the Rho-GAP and tumor suppressor functions of DLC1 by a previously undescribed mechanism. We determined the direct phosphorylation of Y701 of DLC1, which lies in the Rho-GAP domain, by the SRC kinase reduces the binding of Rho-GTP (active Rho) to the Rho-GAP domain of DLC1 and abolishes its Rho-GAP activity. In untransformed and lung adenocarcinoma cell lines, endogenous active SRC and DLC1 co-localized at focal adhesions and formed a protein complex in vivo, implying this interaction is physiologically relevant. The phosphorylation of S129 of DLC1, which lies N-terminal to the Rho-GAP domain, by ERK1 enhanced the binding of the SRC SH3 domain to this region of DLC1 and increased the phosphorylation of Y701 by SRC. These changes reduced the Rho-GAP activity of DLC1, increased Rho-GTP in the cell, and attenuated the DLC1 tumor suppressor functions, as measured by cell migration rate, anchorage-independent growth, and tumor formation in nude mice. Consistent with these observations, mutation of Y701 to F701 increased the Rho-GAP and tumor suppressor activities of DLC1 and decreased Rho-GTP and Rho/ROCK/MRLC signaling. Conversely, mutation of Y701 to the phosphomimetic D701 produced a mutant DLC1 with the opposite phenotype, similar to a ‘GAP-dead’ DLC1 mutant. The Rho-GAP domain of DLC1 was necessary and sufficient for the attenuated Rho-GAP activity attributable to Y701 phosphorylation, as the isolated Rho-GAP domain (residues 609-878) with the Y701F and Y701D mutants displayed, respectively, high and low Rho-GAP activities, as in full-length DLC1. In considering the potential relevance of these findings to human tumors, it is important to recognize that while the Rho-GAP activity of DLC1 is necessary for its full tumor suppressor activity, it is not sufficient, as DLC1 binds several ligands that contribute to this function without attenuating its Rho-GAP activity. Thus, it would be predicted that there would be selective pressure for down-regulation of DLC1 expression even in the presence of high SRC activity. Consistent with this hypothesis, the combination high SRC expression and low DLC1 expression was associated with a poor prognosis in lung adenocarcinomas (p = 0.005) in the TCGA cohort. In summary, the cooperation between the SRC and ERK1 pathways contribute to phosphorylation of Y701, which directly inactivates the Rho-GAP function of DLC1 and attenuates its tumor suppressor activity. These findings are relevant to normal physiology and human cancer. Citation Format: Brajendra K. Tripathi, Xiaolan Qian, Tiera Grant, Philipp Mertins, Dunrui Wang, Alex G. Papageorge, Steven A. Carr, Douglas R. Lowy. Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2155. doi:10.1158/1538-7445.AM2015-2155
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full activity of the deleted in liver cancer 1 dlc1 tumor suppressor depends on an ld like motif that binds talin and focal adhesion kinase fak
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: William C Vass, Alex G Papageorge, Douglas R Lowy, Xiaolan QianAbstract:The deleted in liver cancer 1 (DLC1) tumor suppressor gene, which is frequently inactivated in cancer, encodes a Rho-GAP (GTPase activating protein) focal adhesion protein whose negative regulation of Rho-GTPases is necessary but not sufficient for its full tumor suppressor activity. Here, we report that DLC1 forms a complex with two prooncogenic focal adhesion proteins, talin and the focal adhesion kinase (FAK). We identified an 8-aa sequence (residues 469LDDILYHV476) in DLC1 and designated it an LD-like motif, because it shares homology with the LD motifs of paxillin. This motif was necessary for DLC1 binding to talin and FAK, because a DLC1 mutant, from which six of the residues have been deleted, and another mutant carrying amino acid substitutions in three of the residues are deficient for binding both proteins and localization of DLC1 to focal adhesions. FAK binding was independent of talin and vice versa. In bioassays, both DLC1 mutants were less active than wild-type (WT) DLC1, although the ability of the mutants to negatively regulate overall Rho-GTP was not impaired. We conclude that the LD-like motif, which binds talin and FAK, is required for the full tumor suppressor activity of DLC1 and contributes to the association of DLC1 with focal adhesions.
Fred Etoc - One of the best experts on this subject based on the ideXlab platform.
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DLG1 controls planar spindle orientation in the neuroepithelium through direct interaction with lgn
Journal of Cell Biology, 2014Co-Authors: Mehdi Saadaoui, Mickael Machicoane, Florencia Di Pietro, Fred EtocAbstract:Oriented cell divisions are necessary for the development of epithelial structures. Mitotic spindle orientation requires the precise localization of force generators at the cell cortex via the evolutionarily conserved LGN complex. However, polarity cues acting upstream of this complex in vivo in the vertebrate epithelia remain unknown. In this paper, we show that DLG1 is localized at the basolateral cell cortex during mitosis and is necessary for planar spindle orientation in the chick neuroepithelium. Live imaging revealed that DLG1 is required for directed spindle movements during metaphase. Mechanistically, we show that direct interaction between DLG1 and LGN promotes cortical localization of the LGN complex. Furthermore, in human cells dividing on adhesive micropatterns, homogenously localized DLG1 recruited LGN to the mitotic cortex and was also necessary for proper spindle orientation. We propose that DLG1 acts primarily to recruit LGN to the cortex and that DLG1 localization may additionally provide instructive cues for spindle orientation.
Judy Wai Ping Yam - One of the best experts on this subject based on the ideXlab platform.
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abstract 2141 camp pka signaling enhances activity of deleted in liver cancer 1 dlc1 tumor suppressor in suppressing liver cancer tumorigenesis
Cancer Research, 2012Co-Authors: Lo-kong Chan, Karen Man-fong Sze, Edith Yuk Ting Tse, Yin-shan Yeung, Judy Wai Ping YamAbstract:Deleted in Liver Cancer 1 (DLC1) which encodes a RhoGTPase-activating protein (RhoGAP) is a tumor suppressor frequently inactivated in a wide spectrum of human cancers. The RhoGAP activity has been shown to play a predominant role in the biological activities of DLC1. It has been shown that cells with silenced DLC1 exhibit increased active RhoA level. This finding provides evidence about the activation of RhoA as the consequence of deregulated DLC1 and points to the importance of RhoGAP activity in the biological activity of DLC1. In this regard, it is important to comprehend how RhoGAP activity of DLC1 is related. Here, we show that DLC1 was robustly phosphorylated by cyclic AMP (cAMP)/Protein kinase A (PKA) signaling in cells. Phosphorylation of DLC1 was enhanced by forskolin, a known activator of PKA while suppressed when H-89, an inhibitor of PKA was added. Direct phosphorylation of DLC1 by PKA was further confirmed by the in vitro kinase assay. Using specific phospho-DLC1 antibodies, PKA was shown to phosphorylate DLC1 at S431 and S549. Functional assays demonstrate that phosphorylation at S549 plays a critical role in provoking the inhibitory activity of DLC1 in suppressing growth and motility of Ras-transduced p53 null mouse hepatoblasts. When compared with the stable clone of wild-type DLC1, stable clone of DLC1 phosphomimetic mutant, S549D, displayed a largely reduced growth of subcutaneous and orthotopic liver implanted tumors and an enhanced apoptosis. The migration and invasion rates of S549D cells were also significantly inhibited. Furthermore, S549D expression abolished stress fiber formation but failed to alter filopodia protrusions. These functional effects exerted by S549D were ascribed to the enhanced RhoGAP activity against RhoA. To further investigate the mechanism through which the RhoGAP activity is enhanced, we found that DLC1 dimerized upon S549 phosphorylation. Our findings have revealed for the first time about the regulation of RhoGAP activity of DLC1 via dimerization. Our study suggests a molecular link between PKA and DLC1/Rho pathways and underscores the importance of S549 phosphorylation in the regulation of RhoGAP activity of DLC1. (This study was funded by the Small Project Funding Program, The University of Hong Kong 200907176125) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2141. doi:1538-7445.AM2012-2141
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deleted in liver cancer 1 dlc1 negatively regulates rho rock mlc pathway in hepatocellular carcinoma
PLOS ONE, 2008Co-Authors: Carmen Chak-lui Wong, Yickpang Ching, Chunming Wong, Lo-kong Chan, Judy Wai Ping YamAbstract:Aims Deleted in liver cancer 1 (DLC1), a member of RhoGTPase activating protein (GAP) family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK) is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC). Methodology/Principal Findings We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immumofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2). These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E) abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1) phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage. Conclusion Our data suggest that DLC1 negatively regulates Rho/ROCK/MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma.
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rho gtpase activating protein deleted in liver cancer suppresses cell proliferation and invasion in hepatocellular carcinoma
Cancer Research, 2005Co-Authors: Chunming Wong, Yickpang Ching, Judy Wai Ping Yam, Taion Yau, Thomas Hoyin Leung, Dongyan JinAbstract:Deleted in liver cancer (DLC1) is a candidate tumor suppressor gene recently isolated from human hepatocellular carcinoma. Structurally, DLC1 protein contains a conserved GTPase-activating protein for Rho family protein (RhoGAP) domain, which has been thought to regulate the activity of Rho family proteins. Previous studies indicated that DLC1 was frequently inactivated in cancer cells. In the present study, we aimed to characterize the tumor suppressor roles of DLC1 in hepatocellular carcinoma. We showed that DLC1 significantly inhibited cell proliferation, anchorage-independent growth, and in vivo tumorigenicity when stably expressed in hepatocellular carcinoma cells. Moreover, DLC1 expression greatly reduced the motility and invasiveness of hepatocellular carcinoma cells. With RhoGAP-deficient DLC1 mutant (DLC1-K714E), we showed that the RhoGAP activity was essential for DLC1-mediated tumor suppressor function. Furthermore, the 292– to 648–amino acid region and the steroidogenic acute regulatory related lipid transfer domain played an auxiliary role to RhoGAP and tumor suppressor function of DLC1. Taken together, our findings showed that DLC1 functions as a tumor suppressor in hepatocellular carcinoma and provide the first evidence to support the hypothesis that DLC1 suppresses cancer cell growth by negatively regulating the activity of Rho proteins.
Douglas R Lowy - One of the best experts on this subject based on the ideXlab platform.
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DLC1 deficiency and YAP signaling drive endothelial cell contact inhibition of growth and tumorigenesis
Oncogene, 2019Co-Authors: Lisa Ritchey, Douglas R Lowy, Dunrui Wang, Taekyu Ha, Atsushi Otsuka, Kenji Kabashima, Yuyi Wang, Giovanna TosatoAbstract:Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene deleted in many cancers, including angiosarcoma, an aggressive malignancy of endothelial cell derivation. DLC1-deficiency in primary endothelial cells causes the loss of cell contact inhibition of growth through incompletely defined mechanisms. We report that DLC1 is a regulator of YAP, a transcriptional coactivator of proliferation-promoting and tumor-promoting genes; when confluent, active/nuclear YAP was significantly more abundant in DLC1-deficient endothelial cells compared with control cells. We also found that YAP is a required effector of the loss of cell contact inhibition of growth manifested by DLC1-deficient endothelial cells, as the silencing of YAP prevents this loss. Consistently, human angiosarcomas specimens contained a significantly greater proportion of DLC1^− tumor cells with nuclear YAP compared with the DLC1^+ normal cells in the adjacent tissue. Verteporfin, an inhibitor of YAP, significantly reduced angiosarcoma growth in mice. These results identify YAP as a previously unrecognized effector of DLC1 deficiency-associated loss of cell contact growth inhibition in endothelial cells and a potential therapeutic target in angiosarcoma.
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Abstract 1762: The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1
Molecular and Cellular Biology Genetics, 2019Co-Authors: Beatriz Sanchez-solana, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Parthibane Velayoudame, Jairaj Acharya, Douglas R LowyAbstract:The tumor suppressor gene DLC1 encodes a multi-domain protein, which includes a Rho-GAP domain (Rho GTPase activating protein domain) that negatively regulates the activity of RhoA, B, and C, and has been hypothesized to be the basis of its tumor suppressor functions. DLC1 also contains a less well characterized START (StAR-related lipid transfer) domain in its C-terminus, whose overall contribution to DLC1 function remains incompletely understood. START domains in other proteins have been shown to bind lipids, but no lipid has yet been identified that binds the DLC1 START domain. In the present study, we attempted to gain further insight into the molecular function and lipid binding properties of the DLC1 START domain. We previously determined that Caveolin-1, the main structural component and marker of caveolae, interacts with the DLC1 START domain, and this interaction contributes to the full tumor suppressor activity of DLC1. In unpublished studies, we additionally identified Phospholipase C delta 1 (PLCD1), which participates in intracellular Ca2+ mobilization, forming a complex with the DLC1 START domain and with Caveolin-1. We have now developed evidence that, in addition to binding Caveolin-1 and PLCD1, the DLC1 START domain binds a lipid, phosphatidylserine (PS), and have identified a colon cancer-associated DLC1-START domain mutant (R947C) that results in a protein deficient in forming a complex with all three binding partners and displays reduced tumor suppressor activity but intact RhoGAP activity. The interaction between the DLC1-START domain and PS can occur independently of PLCD1 and/or Caveolin-1, but PS promotes complex formation between the DLC1 START domain and PLCD1 or Caveolin-1, without altering PLCD1 to Caveolin-1 binding. Biological assays with wild type DLC1 show cooperation between DLC1 and PLCD1 or Caveolin-1 to inhibit cell migration, but no cooperation is observed when all three genes are overexpressed, suggesting that Caveolin-1 and PLCD1 occupy the same migration signaling pathway. Thus, we have identified a biologically relevant complex between DLC1 and three macromolecules (PLCD1, PS and Caveolin-1) that contributes to the full tumor suppressor function of DLC1 independently of its RhoGAP activity, and are testing the hypothesis that PS serves as a linker between the DLC1 START domain and PLCD1 or Caveolin-1. Citation Format: Beatriz Sanchez-Solana, Dunrui Wang, Xiaolan Qian, Alex Papageorge, Parthibane Velayoudame, Jairaj Acharya, Douglas R. Lowy. The START domain of the DLC1 tumor suppressor binds phosphatidylserine and two proteins, PLCD1 and Caveolin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1762.
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Abstract 2155: Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma
Cancer Research, 2015Co-Authors: Brajendra K Tripathi, Alex G Papageorge, Xiaolan Qian, Dunrui Wang, Tiera Grant, Philipp Mertins, Steven A. Carr, Douglas R LowyAbstract:The oncogenes SRC and ERK1 are frequently activated in a wide variety of human cancer, while the tumor suppressor DLC1, which encodes a Rho-GAP (GTPase activating protein) essential for its tumor suppressor functions, is frequently down-regulated. However, no prior research has mechanistically linked SRC and ERK1 to DLC1. In this study, we report that SRC and ERK1 cooperate to attenuate the Rho-GAP and tumor suppressor functions of DLC1 by a previously undescribed mechanism. We determined the direct phosphorylation of Y701 of DLC1, which lies in the Rho-GAP domain, by the SRC kinase reduces the binding of Rho-GTP (active Rho) to the Rho-GAP domain of DLC1 and abolishes its Rho-GAP activity. In untransformed and lung adenocarcinoma cell lines, endogenous active SRC and DLC1 co-localized at focal adhesions and formed a protein complex in vivo, implying this interaction is physiologically relevant. The phosphorylation of S129 of DLC1, which lies N-terminal to the Rho-GAP domain, by ERK1 enhanced the binding of the SRC SH3 domain to this region of DLC1 and increased the phosphorylation of Y701 by SRC. These changes reduced the Rho-GAP activity of DLC1, increased Rho-GTP in the cell, and attenuated the DLC1 tumor suppressor functions, as measured by cell migration rate, anchorage-independent growth, and tumor formation in nude mice. Consistent with these observations, mutation of Y701 to F701 increased the Rho-GAP and tumor suppressor activities of DLC1 and decreased Rho-GTP and Rho/ROCK/MRLC signaling. Conversely, mutation of Y701 to the phosphomimetic D701 produced a mutant DLC1 with the opposite phenotype, similar to a ‘GAP-dead’ DLC1 mutant. The Rho-GAP domain of DLC1 was necessary and sufficient for the attenuated Rho-GAP activity attributable to Y701 phosphorylation, as the isolated Rho-GAP domain (residues 609-878) with the Y701F and Y701D mutants displayed, respectively, high and low Rho-GAP activities, as in full-length DLC1. In considering the potential relevance of these findings to human tumors, it is important to recognize that while the Rho-GAP activity of DLC1 is necessary for its full tumor suppressor activity, it is not sufficient, as DLC1 binds several ligands that contribute to this function without attenuating its Rho-GAP activity. Thus, it would be predicted that there would be selective pressure for down-regulation of DLC1 expression even in the presence of high SRC activity. Consistent with this hypothesis, the combination high SRC expression and low DLC1 expression was associated with a poor prognosis in lung adenocarcinomas (p = 0.005) in the TCGA cohort. In summary, the cooperation between the SRC and ERK1 pathways contribute to phosphorylation of Y701, which directly inactivates the Rho-GAP function of DLC1 and attenuates its tumor suppressor activity. These findings are relevant to normal physiology and human cancer. Citation Format: Brajendra K. Tripathi, Xiaolan Qian, Tiera Grant, Philipp Mertins, Dunrui Wang, Alex G. Papageorge, Steven A. Carr, Douglas R. Lowy. Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2155. doi:10.1158/1538-7445.AM2015-2155
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full activity of the deleted in liver cancer 1 dlc1 tumor suppressor depends on an ld like motif that binds talin and focal adhesion kinase fak
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: William C Vass, Alex G Papageorge, Douglas R Lowy, Xiaolan QianAbstract:The deleted in liver cancer 1 (DLC1) tumor suppressor gene, which is frequently inactivated in cancer, encodes a Rho-GAP (GTPase activating protein) focal adhesion protein whose negative regulation of Rho-GTPases is necessary but not sufficient for its full tumor suppressor activity. Here, we report that DLC1 forms a complex with two prooncogenic focal adhesion proteins, talin and the focal adhesion kinase (FAK). We identified an 8-aa sequence (residues 469LDDILYHV476) in DLC1 and designated it an LD-like motif, because it shares homology with the LD motifs of paxillin. This motif was necessary for DLC1 binding to talin and FAK, because a DLC1 mutant, from which six of the residues have been deleted, and another mutant carrying amino acid substitutions in three of the residues are deficient for binding both proteins and localization of DLC1 to focal adhesions. FAK binding was independent of talin and vice versa. In bioassays, both DLC1 mutants were less active than wild-type (WT) DLC1, although the ability of the mutants to negatively regulate overall Rho-GTP was not impaired. We conclude that the LD-like motif, which binds talin and FAK, is required for the full tumor suppressor activity of DLC1 and contributes to the association of DLC1 with focal adhesions.