The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Leslie V Parise - One of the best experts on this subject based on the ideXlab platform.
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cib1 depletion with docetaxel or trail enhances triple negative breast cancer cell death
Cancer Cell International, 2019Co-Authors: Alexander H Chung, Tina M. Leisner, Gabrielle J Dardis, Marissa M Bivins, Alana L Keller, Leslie V PariseAbstract:Patients diagnosed with triple negative breast cancer (TNBC) have limited treatment options and often suffer from resistance and toxicity due to chemotherapy. We previously found that depleting calcium and integrin-binding protein 1 (CIB1) induces cell death selectively in TNBC cells, while sparing normal cells. Therefore, we asked whether CIB1 depletion further enhances tumor-specific killing when combined with either the commonly used chemotherapeutic, docetaxel, or the cell death-inducing ligand, TRAIL. We targeted CIB1 by RNA interference in MDA-MB-436, MDA-MB-231, MDA-MB-468, docetaxel-resistant MDA-MB-436 TNBC cells and ME16C normal breast epithelial cells alone or combination with docetaxel or TRAIL. Cell death was quantified via trypan blue exclusion using flow cytometry and cell death mechanisms were analyzed by Western blotting. Cell surface levels of TRAIL receptors were measured by flow cytometry analysis. CIB1 depletion combined with docetaxel significantly enhanced tumor-specific cell death relative to each treatment alone. The enhanced cell death strongly correlated with caspase-8 activation, a hallmark of death receptor-mediated apoptosis. The death receptor TRAIL-R2 was upregulated in response to CIB1 depletion, which sensitized TNBC cells to the ligand TRAIL, resulting in a synergistic increase in cell death. In addition to death receptor-mediated apoptosis, both combination treatments activated a non-apoptotic mechanism, called paraptosis. Interestingly, these combination treatments also induced nearly complete death of docetaxel-resistant MDA-MB-436 cells, again via apoptosis and paraptosis. In contrast, neither combination treatment induced cell death in normal ME16C cells. Novel combinations of CIB1 depletion with docetaxel or TRAIL selectively enhance naive and docetaxel-resistant TNBC cell death while sparing normal cell. Therefore, combination therapies that target CIB1 could prove to be a safe and durable strategy for treatment of TNBC and potentially other cancers.
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cib1 protects against mptp induced neurotoxicity through inhibiting ask1
Scientific Reports, 2017Co-Authors: Kyoung Wan Yoon, Hyun Suk Yang, Ulhas P Naik, Seong Man Kang, Leslie V Parise, Eui Ju ChoiAbstract:Calcium and integrin binding protein 1 (CIB1) is a calcium-binding protein that was initially identified as a binding partner of platelet integrin αIIb. Although CIB1 has been shown to interact with multiple proteins, its biological function in the brain remains unclear. Here, we show that CIB1 negatively regulates degeneration of dopaminergic neurons in a mouse model of Parkinson’s disease using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Genetic deficiency of the CIB1 gene enhances MPTP-induced neurotoxicity in dopaminergic neurons in CIB1−/− mice. Furthermore, RNAi-mediated depletion of CIB1 in primary dopaminergic neurons potentiated 1-methyl-4-phenyl pyrinidium (MPP+)-induced neuronal death. CIB1 physically associated with apoptosis signal-regulating kinase 1 (ASK1) and thereby inhibited the MPP+-induced stimulation of the ASK1-mediated signaling cascade. These findings suggest that CIB1 plays a protective role in MPTP/MPP+-induced neurotoxicity by blocking ASK1-mediated signaling.
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cib1 a small protein with big ambitions
The FASEB Journal, 2016Co-Authors: Tina M. Leisner, Thomas C Freeman, Justin L. Black, Leslie V PariseAbstract:Calcium- and integrin-binding protein 1 (CIB1) is a small, ubiquitously expressed protein that was first identified as an intracellular binding partner of a platelet-specific α-integrin cytoplasmic tail. Although early studies revealed a role for CIB1 in regulating platelet integrin activity, recent studies have indicated a more diverse role for CIB1 in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. Increasing evidence also points to a novel role for CIB1 in cancer and cardiovascular disease. In addition, an array of CIB1 binding partners has been identified that provide important insight into how CIB1 may regulate these processes. Some of these binding partners include the serine/threonine kinases, p21-activated kinase 1 (PAK1), apoptosis signal-regulating kinase 1 (ASK1), and polo-like kinase 3 (PLK3). Structural and mutational studies indicate that CIB1 binds most or all of its partners via a well-defined hydrophobic cleft. Althou...
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identification of novel integrin binding partners for calcium and integrin binding protein 1 cib1 structural and thermodynamic basis of cib1 promiscuity
Biochemistry, 2013Co-Authors: Thomas C Freeman, Tina M. Leisner, Holly G Bray, Onur Dagliyan, Yi I Wu, Justin L. Black, Ashutosh Tripathy, Nikolay V. Dokholyan, Leslie V PariseAbstract:The short cytoplasmic tails of the α- and β-chains of integrin adhesion receptors regulate integrin activation and cell signaling. Significantly less is known about proteins that bind to α-integrin cytoplasmic tails (CTs) as opposed to β-CTs to regulate integrins. Calcium and integrin binding protein 1 (CIB1) was previously identified as an αIIb binding partner that inhibits agonist-induced activation of the platelet-specific integrin, αIIbβ3. A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site of αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F). Because the CIB1 binding site of αIIb is conserved in all α-integrins and CIB1 expression is ubiquitous, we asked if CIB1 could interact with other α-integrin CTs. We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics. Af...
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the interaction between aid and cib1 is nonessential for antibody gene diversification by gene conversion or class switch recombination
PLOS ONE, 2010Co-Authors: Zachary L Demorest, Donna A Macduff, Scott G Morham, Leslie V Parise, Reuben S. HarrisAbstract:Activation-induced deaminase (AID) initiates somatic hypermutation, gene conversion and class switch recombination by deaminating variable and switch region DNA cytidines to uridines. AID is predominantly cytoplasmic and must enter the nuclear compartment to initiate these distinct antibody gene diversification reactions. Nuclear AID is relatively short-lived, as it is efficiently exported by a CRM1-dependent mechanism and it is susceptible to proteasome-dependent degradation. To help shed light on mechanisms of post-translational regulation, a yeast-based screen was performed to identify AID-interacting proteins. The calcium and integrin binding protein CIB1 was identified by sequencing and the interaction was confirmed by immunoprecipitation experiments. The AID/CIB1 resisted DNase and RNase treatment, and it is therefore unlikely to be mediated by nucleic acid. The requirement for CIB1 in AID-mediated antibody gene diversification reactions was assessed in CIB1-deficient DT40 cells and in knockout mice, but immunoglobulin gene conversion and class switch recombination appeared normal. The DT40 system was also used to show that CIB1 over-expression has no effect on gene conversion and that AID-EGFP subcellular localization is normal. These combined data demonstrate that CIB1 is not required for AID to mediate antibody gene diversification processes. It remains possible that CIB1 has an alternative, a redundant or a subtle non-limiting regulatory role in AID biology.
Ulhas P Naik - One of the best experts on this subject based on the ideXlab platform.
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cib1 protects against mptp induced neurotoxicity through inhibiting ask1
Scientific Reports, 2017Co-Authors: Kyoung Wan Yoon, Hyun Suk Yang, Ulhas P Naik, Seong Man Kang, Leslie V Parise, Eui Ju ChoiAbstract:Calcium and integrin binding protein 1 (CIB1) is a calcium-binding protein that was initially identified as a binding partner of platelet integrin αIIb. Although CIB1 has been shown to interact with multiple proteins, its biological function in the brain remains unclear. Here, we show that CIB1 negatively regulates degeneration of dopaminergic neurons in a mouse model of Parkinson’s disease using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Genetic deficiency of the CIB1 gene enhances MPTP-induced neurotoxicity in dopaminergic neurons in CIB1−/− mice. Furthermore, RNAi-mediated depletion of CIB1 in primary dopaminergic neurons potentiated 1-methyl-4-phenyl pyrinidium (MPP+)-induced neuronal death. CIB1 physically associated with apoptosis signal-regulating kinase 1 (ASK1) and thereby inhibited the MPP+-induced stimulation of the ASK1-mediated signaling cascade. These findings suggest that CIB1 plays a protective role in MPTP/MPP+-induced neurotoxicity by blocking ASK1-mediated signaling.
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binding of cib1 to the αiib tail of αiibβ3 is required for fak recruitment and activation in platelets
PLOS ONE, 2017Co-Authors: Meghna U Naik, Tejal U Naik, Ross Summer, Ulhas P NaikAbstract:Background It is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of αIIb in outside-in signaling is poorly understood. Objectives We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of αIIb and is required for αIIbβ3 outside-in signaling. Here we evaluated the role of CIB1 in regulating outside-in signaling in the absence of inside-out signaling. Methods We used αIIb cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with αIIb subunit as well as Cib1-/- platelets to evaluate the consequence of CIB1 interaction with αIIb on outside-in signaling. Results Fibrinogen binding to αIIbβ3 results in calcium-dependent interaction of CIB1 with αIIb, which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the αIIb complex and its activation. Disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or anti-CIB1 inhibited both FAK association and activation. Furthermore, FAK recruitment to the integrin complex was required for c-Src activation. Inhibition of c-Src had no effect on CIB1 accumulation with the integrin at the filopodia, suggesting that c-Src activity is not required for the formation of CIB1-αIIb-FAK complex. Conclusion Our results suggest that interaction of CIB1 with αIIb is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the αIIbβ3 complex at the filopodia where FAK is activated, which in turn activates c-Src, resulting in propagation of outside-in signaling leading to platelet spreading.
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dual role for cib1 in thrombopoiesis cib1 suppresses megakaryocyte quantities but supports adhesion migration and proplatelet formation
Blood, 2011Co-Authors: John C. Kostyak, Ulhas P NaikAbstract:Abstract 2384 Megakaryocytes (MKs) are large polyploid cells that produce platelets through a process known as thrombopoiesis. Thrombopoietin (Tpo) is the major cytokine that regulates a variety of steps in this process, including hematopoietic stem cell (HSC) differentiation to MKs, proplatelet formation, and platelet release into the circulation. However, the molecular mechanism of thrombopoiesis is poorly understood. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in thrombopoiesis, we utilized a Cib1 −/− mouse model. We observed that Cib1 −/− mice have a slightly elevated number of platelets and bone marrow (BM)-derived MKs than wild-type (WT) controls (p Cib1 −/− and WT mice, suggesting that the defective clearance is not the cause of the observed elevated platelet number. In order to determine if the HSC differentiation is dysregulated by the ablation of Cib1, we analyzed MK-colony forming unit production, which revealed an increase in the colony forming cells with Cib1 deletion compared to WT (p Cib1 −/− mice, cultured with Tpo for 24 hours, produced more highly polyploid MKs than WT BM (p Y925 phosphorylation in Cib1 −/− BM-derived MKs treated with Tpo. Consequently, Akt and ERK1/2 activation downstream of Tpo was enhanced. These results suggested that Cib1 inhibits Tpo signaling by augmenting FAK activation. Interestingly, platelet recovery in Cib1 −/− mice following platelet depletion by experimental immunothrombocytopenia was attenuated compared to WT (p Cib1 −/− MKs compared to WT (p Cib1 −/− MKs formed fewer proplatelets compared to WT (p Disclosures: No relevant conflicts of interest to declare.
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dual role for cib1 in thrombopoiesis cib1 suppresses megakaryocyte quantities but supports adhesion migration and proplatelet formation
Blood, 2011Co-Authors: John C. Kostyak, Ulhas P NaikAbstract:Abstract 2384 Megakaryocytes (MKs) are large polyploid cells that produce platelets through a process known as thrombopoiesis. Thrombopoietin (Tpo) is the major cytokine that regulates a variety of steps in this process, including hematopoietic stem cell (HSC) differentiation to MKs, proplatelet formation, and platelet release into the circulation. However, the molecular mechanism of thrombopoiesis is poorly understood. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in thrombopoiesis, we utilized a Cib1 −/− mouse model. We observed that Cib1 −/− mice have a slightly elevated number of platelets and bone marrow (BM)-derived MKs than wild-type (WT) controls (p Cib1 −/− and WT mice, suggesting that the defective clearance is not the cause of the observed elevated platelet number. In order to determine if the HSC differentiation is dysregulated by the ablation of Cib1, we analyzed MK-colony forming unit production, which revealed an increase in the colony forming cells with Cib1 deletion compared to WT (p Cib1 −/− mice, cultured with Tpo for 24 hours, produced more highly polyploid MKs than WT BM (p Y925 phosphorylation in Cib1 −/− BM-derived MKs treated with Tpo. Consequently, Akt and ERK1/2 activation downstream of Tpo was enhanced. These results suggested that Cib1 inhibits Tpo signaling by augmenting FAK activation. Interestingly, platelet recovery in Cib1 −/− mice following platelet depletion by experimental immunothrombocytopenia was attenuated compared to WT (p Cib1 −/− MKs compared to WT (p Cib1 −/− MKs formed fewer proplatelets compared to WT (p Disclosures: No relevant conflicts of interest to declare.
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calcium dependent inhibition of polo like kinase 3 activity by cib1 in breast cancer cells
International Journal of Cancer, 2011Co-Authors: Meghna U Naik, Ngoc T Pham, Kristin Beebe, Ulhas P NaikAbstract:Members of the polo-like kinases (Plk1, Plk2, Plk3, and Plk4) are involved in the regulation of various stages of the cell cycle and have been implicated in cancer progression. Unlike its other family members the expression of Plk3 remains steady during cell cycle progression, suggesting that its activity may be spatiotemporally regulated. However, the mechanism of regulation of Plk3 activity is not well understood. Here, we show that calcium- and integrin-binding protein 1 (CIB1), a Plk3 interacting protein, is widely expressed in various cancer cell lines. Expression of CIB1 mRNA as well as protein is increased in breast cancer tissue as compared to normal tissue. CIB1 constitutively interacts with Plk3 as determined by both in vitro and in vivo assays. This interaction of CIB1 with Plk3 is independent of intracellular Ca 2+ . Furthermore, binding of CIB1 results in inhibition of Plk3 kinase activity both in vitro and in vivo. Interestingly, this inhibition of the Plk3 activity by CIB1 is Ca 2+ -dependent. Taken together, our results suggest that CIB1 is a regulatory subunit of Plk3 and it regulates Plk3 activity in a Ca 2+ -dependent manner. Furthermore, upregulation of CIB1 in cancer cells could thus inhibit Plk3 activity leading to abnormal cell cycle regulation in breast cancer cells. Thus in addition to Plk3, CIB1 may be a potential biomarker and target for therapeutic intervention of breast cancer.
Meghna U Naik - One of the best experts on this subject based on the ideXlab platform.
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binding of cib1 to the αiib tail of αiibβ3 is required for fak recruitment and activation in platelets
PLOS ONE, 2017Co-Authors: Meghna U Naik, Tejal U Naik, Ross Summer, Ulhas P NaikAbstract:Background It is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of αIIb in outside-in signaling is poorly understood. Objectives We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of αIIb and is required for αIIbβ3 outside-in signaling. Here we evaluated the role of CIB1 in regulating outside-in signaling in the absence of inside-out signaling. Methods We used αIIb cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with αIIb subunit as well as Cib1-/- platelets to evaluate the consequence of CIB1 interaction with αIIb on outside-in signaling. Results Fibrinogen binding to αIIbβ3 results in calcium-dependent interaction of CIB1 with αIIb, which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the αIIb complex and its activation. Disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or anti-CIB1 inhibited both FAK association and activation. Furthermore, FAK recruitment to the integrin complex was required for c-Src activation. Inhibition of c-Src had no effect on CIB1 accumulation with the integrin at the filopodia, suggesting that c-Src activity is not required for the formation of CIB1-αIIb-FAK complex. Conclusion Our results suggest that interaction of CIB1 with αIIb is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the αIIbβ3 complex at the filopodia where FAK is activated, which in turn activates c-Src, resulting in propagation of outside-in signaling leading to platelet spreading.
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calcium dependent inhibition of polo like kinase 3 activity by cib1 in breast cancer cells
International Journal of Cancer, 2011Co-Authors: Meghna U Naik, Ngoc T Pham, Kristin Beebe, Ulhas P NaikAbstract:Members of the polo-like kinases (Plk1, Plk2, Plk3, and Plk4) are involved in the regulation of various stages of the cell cycle and have been implicated in cancer progression. Unlike its other family members the expression of Plk3 remains steady during cell cycle progression, suggesting that its activity may be spatiotemporally regulated. However, the mechanism of regulation of Plk3 activity is not well understood. Here, we show that calcium- and integrin-binding protein 1 (CIB1), a Plk3 interacting protein, is widely expressed in various cancer cell lines. Expression of CIB1 mRNA as well as protein is increased in breast cancer tissue as compared to normal tissue. CIB1 constitutively interacts with Plk3 as determined by both in vitro and in vivo assays. This interaction of CIB1 with Plk3 is independent of intracellular Ca 2+ . Furthermore, binding of CIB1 results in inhibition of Plk3 kinase activity both in vitro and in vivo. Interestingly, this inhibition of the Plk3 activity by CIB1 is Ca 2+ -dependent. Taken together, our results suggest that CIB1 is a regulatory subunit of Plk3 and it regulates Plk3 activity in a Ca 2+ -dependent manner. Furthermore, upregulation of CIB1 in cancer cells could thus inhibit Plk3 activity leading to abnormal cell cycle regulation in breast cancer cells. Thus in addition to Plk3, CIB1 may be a potential biomarker and target for therapeutic intervention of breast cancer.
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cib1 regulates thrombosis through integrin αaiibβ3 outside in signaling but not inside out signaling
Blood, 2009Co-Authors: Meghna U Naik, Ulhas P NaikAbstract:Abstract 2992 Poster Board II-969 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out that leads to the activation of integrin αaIIbβ3. Upon ligand binding to integrin αaIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. The regulation of these signaling events is not well understood. Previously, we had identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with integrin αaIIbβ3. Using a novel technique to inhibit interaction of CIB1 with integrin αaIIbβ3 in intact platelets, we have shown that CIB1 regulates outside-in signaling through integrin αaIIbβ3. Recently, using Cib1-/- mice, we confirmed that CIB1 is a key regulator of hemostasis. FeCl3-induced carotid artery thrombosis, a well recognized in vivo model of thrombosis showed a significantly extended time of occlusion in Cib1-/- mice compared to wild type (WT) mice. Since CIB1 is expressed both in the endothelium and in platelets, the observed defect could arise from the lack of CIB1 in either cell types. To identify the specific involvement of platelets, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs due to platelet thrombus formation without injury to the endothelium. We found no difference in the number of Cib1-/- mice and WT mice that died within two minutes (n=20). When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, no difference between Cib1-/- and WT mice was observed. Consistent with this finding, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αaIIbβ3 was also not affected by Cib1 deficiency. When we analyzed the rate of clot retraction, we found that incomplete and significantly (P Disclosures: No relevant conflicts of interest to declare.
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cib1 regulates thrombosis through integrin αaiibβ3 outside in signaling but not inside out signaling
Blood, 2009Co-Authors: Meghna U Naik, Ulhas P NaikAbstract:Abstract 2992 Poster Board II-969 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out that leads to the activation of integrin αaIIbβ3. Upon ligand binding to integrin αaIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. The regulation of these signaling events is not well understood. Previously, we had identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with integrin αaIIbβ3. Using a novel technique to inhibit interaction of CIB1 with integrin αaIIbβ3 in intact platelets, we have shown that CIB1 regulates outside-in signaling through integrin αaIIbβ3. Recently, using Cib1-/- mice, we confirmed that CIB1 is a key regulator of hemostasis. FeCl3-induced carotid artery thrombosis, a well recognized in vivo model of thrombosis showed a significantly extended time of occlusion in Cib1-/- mice compared to wild type (WT) mice. Since CIB1 is expressed both in the endothelium and in platelets, the observed defect could arise from the lack of CIB1 in either cell types. To identify the specific involvement of platelets, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs due to platelet thrombus formation without injury to the endothelium. We found no difference in the number of Cib1-/- mice and WT mice that died within two minutes (n=20). When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, no difference between Cib1-/- and WT mice was observed. Consistent with this finding, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αaIIbβ3 was also not affected by Cib1 deficiency. When we analyzed the rate of clot retraction, we found that incomplete and significantly (P Disclosures: No relevant conflicts of interest to declare.
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cib1 deficiency results in impaired thrombosis the potential role of cib1 in outside in signaling through integrin αiibβ3
Journal of Thrombosis and Haemostasis, 2009Co-Authors: Meghna U Naik, Padmini Manrai, Peter G Millili, Millicent O Sullivan, A. Nigam, Kirk J Czymmek, Ulhas P NaikAbstract:Summary. Background: Agonist-induced inside-out signaling activates platelet integrin αIIbβ3, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through αIIbβ3 regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating αIIbβ3 function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl3-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin β3 subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.
Hans J Vogel - One of the best experts on this subject based on the ideXlab platform.
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solution structures of ca2 cib1 and mg2 cib1 and their interactions with the platelet integrin αiib cytoplasmic domain
Journal of Biological Chemistry, 2011Co-Authors: Hao Huang, Hiroaki Ishida, Aaron P. Yamniuk, Hans J VogelAbstract:The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca2+-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca2+-CIB1 and Mg2+-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca2+-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca2+-CIB1 and Mg2+-CIB1 have similar structures, but the N-lobe of Mg2+-CIB1 is slightly more opened than that of Ca2+-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca2+-CIB1 and Mg2+-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca2+-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca2+-CIB1 and Mg2+-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.
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auxiliary ca2 binding sites can influence the structure of cib1
Protein Science, 2009Co-Authors: Aaron P. Yamniuk, Kathryn L Anderson, Marie E Fraser, Hans J VogelAbstract:Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca2+ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (Kd, 10−2M) Ca2+ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca2+ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca2+ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca2+ concentrations.
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auxiliary ca2 binding sites can influence the structure of cib1
Protein Science, 2009Co-Authors: Aaron P. Yamniuk, Kathryn L Anderson, Marie E Fraser, Hans J VogelAbstract:Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca2+ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (Kd, 10−2M) Ca2+ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca2+ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca2+ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca2+ concentrations.
Hartwig Schmale - One of the best experts on this subject based on the ideXlab platform.
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sweet taste receptor interacting protein cib1 is a general inhibitor of insp3 dependent ca2 release in vivo
Journal of Neurochemistry, 2008Co-Authors: Jan K Hennigs, Nicole Burhenne, Frauke Stähler, Marcel Winnig, Bettina Walter, Wolfgang Meyerhof, Hartwig SchmaleAbstract:In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα16gust44 and Gα15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca2+ concentration, we used sweet and umami compounds as well as other InsP3-generating ligands in FURA-2-based Ca2+ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca2+ response of HEK293 cells to the InsP3-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca2+ store of the endoplasmic reticulum (ER) and independent of extracellular Ca2+. The function of CIB1 on InsP3-evoked Ca2+ release from the ER is most likely mediated by its interaction with the InsP3 receptor. Thus, CIB1 seems to be an inhibitor of InsP3-dependent Ca2+ release in vivo.
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sweet taste receptor interacting protein cib1 is a general inhibitor of insp3 dependent ca2 release in vivo
Journal of Neurochemistry, 2008Co-Authors: Jan K Hennigs, Nicole Burhenne, Frauke Stähler, Marcel Winnig, Bettina Walter, Wolfgang Meyerhof, Hartwig SchmaleAbstract:In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα16gust44 and Gα15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca2+ concentration, we used sweet and umami compounds as well as other InsP3-generating ligands in FURA-2-based Ca2+ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca2+ response of HEK293 cells to the InsP3-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca2+ store of the endoplasmic reticulum (ER) and independent of extracellular Ca2+. The function of CIB1 on InsP3-evoked Ca2+ release from the ER is most likely mediated by its interaction with the InsP3 receptor. Thus, CIB1 seems to be an inhibitor of InsP3-dependent Ca2+ release in vivo.