Multiple studies indicate that iron chelators enhance their anti-cancer properties by inducing ND... more Multiple studies indicate that iron chelators enhance their anti-cancer properties by inducing NDRG1, a known tumor and metastasis suppressor. However, the exact role of NDRG1 remains controversial, as newer studies have shown that NDRG1 can also act as an oncogene. Our group recently introduced mitochondrially targeted iron chelators deferoxamine (mitoDFO) and deferasirox (mitoDFX) as effective anti-cancer agents. In this study, we evaluated the ability of these modified chelators to induce NDRG1 and the role of NDRG1 in breast cancer. We demonstrated that both compounds specifically increase NDRG1 without inducing other NDRG family members. We have documented that the effect of mitochondrially targeted chelators is at least partially mediated by GSK3α/β, leading to phosphorylation of NDRG1 at Thr 346 and to a lesser extent on Ser 330. Loss of NDRG1 increases cell death induced by mitoDFX. Notably, MDA-MB-231 cells lacking NDRG1 exhibit reduced extracellular acidification rate and grow slower than parental cells, while the opposite is true for ER+ MCF7 cells. Moreover, overexpression of full-length NDRG1 and the N-terminally truncated isoform (59112) significantly reduced sensitivity towards mitoDFX in ER+ cells. Furthermore, cells overexpressing fulllength NDRG1 exhibited a significantly accelerated tumor formation, while its N-terminally truncated isoforms showed significantly impaired capacity to form tumors. Thus, overexpression of full-length NDRG1 promotes tumor growth in highly aggressive triple-negative breast cancer.
The International Journal of Developmental Biology, 2019
Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. ... more Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation, whereas, mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We also review functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments have suggested novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions.
Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is... more Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.
The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). ... more The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients and the cellular energy state; TORC2 regulates AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture (phagocytosis) in Dictyostelium is independent of TORC1-mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3 and Lst8 promotes nutrient particle uptake; inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either of the TOR complexes. The integrated and balanced regulation of TORC1 and TORC2 might be crucial in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes.
Highlights d FRET-based Src biosensor was developed and functionally verified d Src activatory mu... more Highlights d FRET-based Src biosensor was developed and functionally verified d Src activatory mutations induce opening of Src structure d Src inhibitors differentially affect Src structure and its cellular localization d Src activity is high in FAs and increases/decreases during FA assembly/disassembly
Deferoxamine (DFO) represents a widely used iron chelator for the treatment of iron overload. Her... more Deferoxamine (DFO) represents a widely used iron chelator for the treatment of iron overload. Here we describe the use of mitochondrially targeted deferoxamine (mitoDFO) as a novel approach to preferentially target cancer cells. The agent showed marked cytostatic, cytotoxic, and migrastatic properties in vitro, and it significantly suppressed tumor growth and metastasis in vivo. The underlying molecular mechanisms included (i) impairment of iron-sulfur [Fe-S] cluster/heme biogenesis, leading to destabilization and loss of activity of [Fe-S] cluster/heme containing enzymes, (ii) inhibition of mitochondrial respiration leading to mitochondrial reactive oxygen species production, resulting in dysfunctional mitochondria with markedly reduced supercomplexes, and (iii) fragmentation of the mitochondrial network and induction of mitophagy. Mitochondrial targeting of deferoxamine represents a way to deprive cancer cells of biologically active iron, which is incompatible with their prolifera...
MT1-MMP (MMP-14) is a multifunctional protease that regulates ECM degradation, activation of othe... more MT1-MMP (MMP-14) is a multifunctional protease that regulates ECM degradation, activation of other proteases, and a variety of cellular processes, including migration and viability in physiological and pathological contexts. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain that constitutes the final 20 C-terminal amino acids, while the rest of the protease is extracellular. In this review, we summarize the ways in which the cytoplasmic tail is involved in regulating and enacting the functions of MT1-MMP. We also provide an overview of known interactors of the MT1-MMP cytoplasmic tail and the functional significance of these interactions, as well as further insight into the mechanisms of cellular adhesion and invasion that are regulated by the cytoplasmic tail.
The FDA-approved drugs raloxifene and bazedoxifene could be among the best candidates to prevent ... more The FDA-approved drugs raloxifene and bazedoxifene could be among the best candidates to prevent mortality in severe COVID-19 patients. Raloxifene and bazedoxifene inhibit IL-6 signaling at therapeutic doses, suggesting they have the potential to prevent the cytokine storm, ARDS and mortality in severe COVID-19 patients, as is being shown with humanized antibodies blocking IL-6 signaling. In addition, raloxifene and bazedoxifene are selective estrogen receptor modulators with strong antiviral activity.
The concept of 'migrastatics' allows the development of a new drug class that is neither ... more The concept of 'migrastatics' allows the development of a new drug class that is neither cytotoxic nor antiproliferative but is solely directed towards inhibition of cancer cell motility. Given that the regulatory pathway is open, and migrastatic candidates have been described, it is the right time to enter a new era of antimetastatic treatment.
bioRxiv (Cold Spring Harbor Laboratory), Aug 24, 2022
The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interac... more The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signaling hub in a variety of cellular processes.
Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by... more Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligandbinding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/-mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells. INTRODUCTION Crk-associated substrate (CAS) is a major Src substrate implicated in integrin control of cell behavior (reviewed in Defilippi et al., 2006). Reexpression of CAS in cas-deficient mouse embryo fibroblasts transformed by oncogenic Src promotes enhanced cell invasiveness, reorganization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins focal adhesion kinase (FAK) and paxillin (Brabek et al., 2004). Moreover, CAS signaling is implicated in lung metastasis of Src-transformed mouse embryo fibroblasts (Brabek et al., 2005). The human orthologue of CAS (termed BCAR1, for breast cancer antiestrogen resistance) was identified in a functional screen for genes involved in resistance of breast cancer cells to antiestrogenic drugs (Brinkman et al., 2000). In breast cancer patients, high CAS/BCAR1 levels are associated with early disease recurrence, poor response to tamoxifen treatment, and lower overall survival (Dorssers et al., 2004). Structurally, CAS is composed of an N-terminal Src homology 3 (SH3) domain that binds to FAK and a C-terminal Src-binding domain (SBD) that includes a binding site for the SH3 domain of Src family of kinases (SFKs; Sakai et al., 1994). The central region of CAS contains a substrate domain (SD), characterized by 15 Tyr-X-X-Pro (YxxP) motifs. SFKs, either directly bound to the CAS SBD or indirectly associated with CAS through a FAK bridge, appear to phosphorylate many or all of the CAS SD YxxP tyrosines (Ruest et al., 2001; Shin et al., 2004).
In this review protocols are described for studying protein tyrosine kinase signalling upon integ... more In this review protocols are described for studying protein tyrosine kinase signalling upon integrin-mediated cell adhesion. We have outlined detailed procedures for fibronectin-replating experiment, biochemical examination of the phosphotyrosine content of cellular proteins by immunoblotting using phosphorylation-specific antibodies or immunoprecipitation and analysis with general phosphotyrosine antibodies. Despite great advances that were made toward optimizing the described procedures, all these methods still remain in many respects an art, given the plentiful of variables and the extent to which the optimum conditions vary from one experimental condition to the other. Examples of performed experiments using the described procedures thus also include notes regarding variability of approaches based on experimental conditions.
bioRxiv (Cold Spring Harbor Laboratory), May 30, 2018
Gemperle et al. present the first report of an interaction between p130Cas with the serine/threon... more Gemperle et al. present the first report of an interaction between p130Cas with the serine/threonine kinase PKN3, implicated in prostate and breast cancer growth. They show that p130Cas colocalizes with PKN3 in cell structures that have a pro-invasive function and enhance our understanding of PKN3-mediated signaling and tumor growth.
G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress an... more G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks' Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated. SCANNING 31: 102-106, 2009.
Multiple studies indicate that iron chelators enhance their anti-cancer properties by inducing ND... more Multiple studies indicate that iron chelators enhance their anti-cancer properties by inducing NDRG1, a known tumor and metastasis suppressor. However, the exact role of NDRG1 remains controversial, as newer studies have shown that NDRG1 can also act as an oncogene. Our group recently introduced mitochondrially targeted iron chelators deferoxamine (mitoDFO) and deferasirox (mitoDFX) as effective anti-cancer agents. In this study, we evaluated the ability of these modified chelators to induce NDRG1 and the role of NDRG1 in breast cancer. We demonstrated that both compounds specifically increase NDRG1 without inducing other NDRG family members. We have documented that the effect of mitochondrially targeted chelators is at least partially mediated by GSK3α/β, leading to phosphorylation of NDRG1 at Thr 346 and to a lesser extent on Ser 330. Loss of NDRG1 increases cell death induced by mitoDFX. Notably, MDA-MB-231 cells lacking NDRG1 exhibit reduced extracellular acidification rate and grow slower than parental cells, while the opposite is true for ER+ MCF7 cells. Moreover, overexpression of full-length NDRG1 and the N-terminally truncated isoform (59112) significantly reduced sensitivity towards mitoDFX in ER+ cells. Furthermore, cells overexpressing fulllength NDRG1 exhibited a significantly accelerated tumor formation, while its N-terminally truncated isoforms showed significantly impaired capacity to form tumors. Thus, overexpression of full-length NDRG1 promotes tumor growth in highly aggressive triple-negative breast cancer.
The International Journal of Developmental Biology, 2019
Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. ... more Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation, whereas, mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We also review functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments have suggested novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions.
Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is... more Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.
The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). ... more The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients and the cellular energy state; TORC2 regulates AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture (phagocytosis) in Dictyostelium is independent of TORC1-mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3 and Lst8 promotes nutrient particle uptake; inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either of the TOR complexes. The integrated and balanced regulation of TORC1 and TORC2 might be crucial in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes.
Highlights d FRET-based Src biosensor was developed and functionally verified d Src activatory mu... more Highlights d FRET-based Src biosensor was developed and functionally verified d Src activatory mutations induce opening of Src structure d Src inhibitors differentially affect Src structure and its cellular localization d Src activity is high in FAs and increases/decreases during FA assembly/disassembly
Deferoxamine (DFO) represents a widely used iron chelator for the treatment of iron overload. Her... more Deferoxamine (DFO) represents a widely used iron chelator for the treatment of iron overload. Here we describe the use of mitochondrially targeted deferoxamine (mitoDFO) as a novel approach to preferentially target cancer cells. The agent showed marked cytostatic, cytotoxic, and migrastatic properties in vitro, and it significantly suppressed tumor growth and metastasis in vivo. The underlying molecular mechanisms included (i) impairment of iron-sulfur [Fe-S] cluster/heme biogenesis, leading to destabilization and loss of activity of [Fe-S] cluster/heme containing enzymes, (ii) inhibition of mitochondrial respiration leading to mitochondrial reactive oxygen species production, resulting in dysfunctional mitochondria with markedly reduced supercomplexes, and (iii) fragmentation of the mitochondrial network and induction of mitophagy. Mitochondrial targeting of deferoxamine represents a way to deprive cancer cells of biologically active iron, which is incompatible with their prolifera...
MT1-MMP (MMP-14) is a multifunctional protease that regulates ECM degradation, activation of othe... more MT1-MMP (MMP-14) is a multifunctional protease that regulates ECM degradation, activation of other proteases, and a variety of cellular processes, including migration and viability in physiological and pathological contexts. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain that constitutes the final 20 C-terminal amino acids, while the rest of the protease is extracellular. In this review, we summarize the ways in which the cytoplasmic tail is involved in regulating and enacting the functions of MT1-MMP. We also provide an overview of known interactors of the MT1-MMP cytoplasmic tail and the functional significance of these interactions, as well as further insight into the mechanisms of cellular adhesion and invasion that are regulated by the cytoplasmic tail.
The FDA-approved drugs raloxifene and bazedoxifene could be among the best candidates to prevent ... more The FDA-approved drugs raloxifene and bazedoxifene could be among the best candidates to prevent mortality in severe COVID-19 patients. Raloxifene and bazedoxifene inhibit IL-6 signaling at therapeutic doses, suggesting they have the potential to prevent the cytokine storm, ARDS and mortality in severe COVID-19 patients, as is being shown with humanized antibodies blocking IL-6 signaling. In addition, raloxifene and bazedoxifene are selective estrogen receptor modulators with strong antiviral activity.
The concept of 'migrastatics' allows the development of a new drug class that is neither ... more The concept of 'migrastatics' allows the development of a new drug class that is neither cytotoxic nor antiproliferative but is solely directed towards inhibition of cancer cell motility. Given that the regulatory pathway is open, and migrastatic candidates have been described, it is the right time to enter a new era of antimetastatic treatment.
bioRxiv (Cold Spring Harbor Laboratory), Aug 24, 2022
The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interac... more The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signaling hub in a variety of cellular processes.
Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by... more Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligandbinding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein-tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas-/-mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells. INTRODUCTION Crk-associated substrate (CAS) is a major Src substrate implicated in integrin control of cell behavior (reviewed in Defilippi et al., 2006). Reexpression of CAS in cas-deficient mouse embryo fibroblasts transformed by oncogenic Src promotes enhanced cell invasiveness, reorganization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins focal adhesion kinase (FAK) and paxillin (Brabek et al., 2004). Moreover, CAS signaling is implicated in lung metastasis of Src-transformed mouse embryo fibroblasts (Brabek et al., 2005). The human orthologue of CAS (termed BCAR1, for breast cancer antiestrogen resistance) was identified in a functional screen for genes involved in resistance of breast cancer cells to antiestrogenic drugs (Brinkman et al., 2000). In breast cancer patients, high CAS/BCAR1 levels are associated with early disease recurrence, poor response to tamoxifen treatment, and lower overall survival (Dorssers et al., 2004). Structurally, CAS is composed of an N-terminal Src homology 3 (SH3) domain that binds to FAK and a C-terminal Src-binding domain (SBD) that includes a binding site for the SH3 domain of Src family of kinases (SFKs; Sakai et al., 1994). The central region of CAS contains a substrate domain (SD), characterized by 15 Tyr-X-X-Pro (YxxP) motifs. SFKs, either directly bound to the CAS SBD or indirectly associated with CAS through a FAK bridge, appear to phosphorylate many or all of the CAS SD YxxP tyrosines (Ruest et al., 2001; Shin et al., 2004).
In this review protocols are described for studying protein tyrosine kinase signalling upon integ... more In this review protocols are described for studying protein tyrosine kinase signalling upon integrin-mediated cell adhesion. We have outlined detailed procedures for fibronectin-replating experiment, biochemical examination of the phosphotyrosine content of cellular proteins by immunoblotting using phosphorylation-specific antibodies or immunoprecipitation and analysis with general phosphotyrosine antibodies. Despite great advances that were made toward optimizing the described procedures, all these methods still remain in many respects an art, given the plentiful of variables and the extent to which the optimum conditions vary from one experimental condition to the other. Examples of performed experiments using the described procedures thus also include notes regarding variability of approaches based on experimental conditions.
bioRxiv (Cold Spring Harbor Laboratory), May 30, 2018
Gemperle et al. present the first report of an interaction between p130Cas with the serine/threon... more Gemperle et al. present the first report of an interaction between p130Cas with the serine/threonine kinase PKN3, implicated in prostate and breast cancer growth. They show that p130Cas colocalizes with PKN3 in cell structures that have a pro-invasive function and enhance our understanding of PKN3-mediated signaling and tumor growth.
G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress an... more G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks' Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated. SCANNING 31: 102-106, 2009.
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Papers by Daniel Rösel