Calcium And Integrin-binding Protein 1 Research Articles

Identification of calcium and integrin-binding protein 1 as a reprogrammed glucose metabolism mediator to restrict immune cell infiltration in the stromal compartment of pancreatic ductal adenocarcinoma.

An increasing body of evidence has suggested that reprogrammed metabolism plays a critical role in the progression of pancreatic ductal adenocarcinoma (PDAC) by affecting the tumor and stromal cellular components in the tumor microenvironment (TME). By analyzing the KRAS pathway and metabolic pathways, we found that calcium and integrin-binding protein 1 (CIB1) corresponded with upregulation of glucose metabolism pathways and was associated with poor prognosis in patients with PDAC from The Cancer Genome Atlas (TCGA). Elevated CIB1 expression combined with upregulated glycolysis, oxidative phosphorylation (Oxphos), hypoxia pathway activation, and cell cycle promoted PDAC tumor growth and increased tumor cellular com-ponents. Furthermore, we confirmed the mRNA overexpression of CIB1 and co-expression of CIB1 and KRAS mutation in cell lines from the Expression Atlas. Subsequently, immunohistochemistry staining from the Human Protein Atlas (HPA) showed that high expression of CIB1 in tumor cells was associated with an increased tumor compartment and reduced stromal cellular abundance. Furthermore, using multiplexed immunohistochemistry (mIHC), we verified that low stromal abundance was correlated with low infiltration of CD8+ PD-1- T cells which led to suppressed anti-tumor immunity. Overall, our findings identify CIB1 as a metabolic pathway-mediated factor for the restriction of immune cell infiltration in the stromal compartment of PDAC and highlight the potential value of CIB1 as a prognostic biomarker involved in metabolic reprogramming and immune modulation.

Identification of novel regulators involved in AD pathogenesis using the CRISPR-Cas9 system

The production of amyloid β peptide (Aβ) is an important process relating to the pathogenesis of Alzheimer disease (AD). It is widely known that the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases lead to the production of Aβ. However, the precise regulatory mechanism for Aβ production remains unclear. We have established a CRISPR-Cas9 based screening system to identify the novel regulators of Aβ production. Calcium and integrin-binding protein 1 (CIB1) was identified as a novel potential negative regulator of Aβ production. The knockdown and knockout of Cib1 significantly increased Aβ levels. In addition, immunoprecipitation showed that CIB1 interacts with the γ-secretase complex but did not alter its enzymatic activity. Moreover, Cib1 disruption specifically reduced the cell-surface localization of the γ-secretase complex. Finally, the single-cell RNA-seq analysis in the human brain demonstrated that early-stage AD patients have lower neuronal CIB1 mRNA levels compared to healthy controls. Taken together, we have shown that CIB1 controls the subcellular localization of γ-secretase, resulting in the regulation of Aβ production, suggesting the involvement of CIB1 in the development of AD pathogenesis.

Roles of Focal Adhesion Kinase PTK2 and Integrin αIIbβ3 Signaling in Collagen- and GPVI-Dependent Thrombus Formation under Shear.

Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling.

An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins

The PKC family consists of several closely related kinases. These enzymes regulate the function of proteins through the phosphorylation of hydroxyl groups on serines and/or threonines. The selective activation of individual PKC isozymes has proven challenging because of a lack of specific activator molecules. Here, we developed an optogenetic blue light–activated PKC isozyme that harnesses a plant-based dimerization system between the photosensitive cryptochrome-2 (CRY2) and the N terminus of the transcription factor calcium and integrin-binding protein 1 (CIB1) (N-terminal region of the CRY2-binding domain of CIB1). We show that tagging CRY2 with the catalytic domain of PKC isozymes can efficiently promote its translocation to the cell surface upon blue light exposure. We demonstrate this system using PKCε and show that this leads to robust activation of a K+ channel (G protein–gated inwardly rectifying K+ channels 1 and 4), previously shown to be activated by PKCε. We anticipate that this approach can be utilized for other PKC isoforms to provide a reliable and direct stimulus for targeted membrane protein phosphorylation by the relevant PKCs.

Calcium and integrin-binding protein 1-like interacting with an integrin α-cytoplasmic domain facilitates cellular immunity in Helicoverpa armigera

Integrins are transmembrane receptor heterodimers composed of α and β subunits. They are known to mediate extracellular signals to promote cell adhesion and spreading, and are therefore essential for cellular immunity. However, proteins that bind to integrin cytoplasmic domains and mediate intracellular signaling to promote cell adhesion require identification. Calcium and integrin-binding protein 1 (CIB1) that binds to the integrin α-cytoplasmic domain has rarely been examined in insects. In this study, we found that 20-hydroxyecdysone promoted cell phagocytosis and spreading in Helicoverpa armigera. Transcriptomic analyses of hemocytes identified an integrin α gene (HaINTα-PS1) whose expression could be induced by either 20-hydroxyecdysone injection or bead challenge. Isothermal titration calorimetry assays showed that H. armigera CIB1-like (HaCIB1-like) weakly bound to the cytoplasmic domain of HaINTα-PS1 in the presence of calcium. HaINTα-PS1 or HaCIB1-like knockdown inhibited hemocytic encapsulation and phagocytosis, and plasmatocyte spreading. Moreover, HaCIB1-like overexpression in a H. armigera epidermal cell line overexpanded cells and impaired cell phagocytosis. Thus, insect CIB1-like potentially interacted with integrin α-cytoplasmic domain and facilitated cell adhesion. This study enriches our understanding of the molecular mechanism underlying integrin-mediated cellular immunity in insects.

The Relationship of Sphingosine Kinase 1 With Pyroptosis Provides a New Strategy for Tumor Therapy.

Sphingosine kinase 1 (SPHK1) is a crucial molecule that catalyzes sphingosine to synthesize sphingosine-1-phosphate (S1P), facilitating cell survival signaling. Pyroptosis is a perplexing inflammatory mode of cell death primarily triggered by caspase-1, evoked by the NLRP3 inflammasome. Sphingosine is identified as a danger-associated molecular pattern (DAMP), which activates the NLRP3 inflammasome assembly and induces the pyroptosis. It has been demonstrated that macrophages play a pro-tumorigenic role and are closely associated with tumor progression. Attenuation of SPHK1 activity contributes significantly to macrophage pyroptosis and tumor inhibition. Calcium and integrin-binding protein 1 (CIB1) plays an important role in the translocation of SPHK1 from the cytoplasm to the plasma membrane, whereas CIB2 blocks the subcellular trafficking of SPHK1. Therefore, knockout of CIB1 or over-expression of CIB2 will result in sphingosine accumulation and contribute significantly to cancer treatment by several approaches. First, it directly provokes cancer cell apoptosis or triggers robust anti-tumor immunity by pyroptosis-induced inflammation. Second, it could restrain SPHK1 translocation from the cytoplasm to the plasma membrane and further pyroptosis, which not only drive M2 macrophages death but also facilitate tumor microenvironment inflammation as well as the further release of sphingosine from damaged macrophages. The perspective might provide novel insight into the association between SPHK1 and pyroptosis and suggest the potential target for cancer therapy.

Identification of calcium and integrin-binding protein 1 as a novel regulator of production of amyloid β peptide using CRISPR/Cas9-based screening system.

The aberrant metabolism of amyloid β peptide (Aβ) has been implicated in the etiology of Alzheimer disease (AD). Aβ is produced via the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. However, the precise regulatory mechanism of Aβ generation still remains unclear. To gain a better understanding of the molecular mechanism of Aβ production, we established a genetic screening method based on the CRISPR/Cas9 system to identify novel regulators of Aβ production. We successfully identified calcium and integrin-binding protein 1 (CIB1) as a potential negative regulator of Aβ production. The disruption of Cib1 significantly upregulated Aβ levels. In addition, immunoprecipitation experiments demonstrated that CIB1 interacts with the γ-secretase complex. Moreover, the disruption of Cib1 specifically reduced the cell-surface localization of mature Nicastrin (Nct), which is a component of the γ-secretase complex, without changing the intrinsic activity of γ-secretase. Finally, we confirmed using the single-cell RNA-seq data in human that CIB1 mRNA level in neuron was decreased in the early stage of AD. Taken together, our results indicate that CIB1 regulates Aβ production via controlling the subcellular localization of γ-secretase, suggesting CIB1 is involved in the development of AD.

Integration of transcriptome-wide association study and messenger RNA expression profile to identify genes associated with osteoarthritis.

AimsOsteoarthritis (OA) is the most prevalent joint disease. However, the specific and definitive genetic mechanisms of OA are still unclear.MethodsTissue-related transcriptome-wide association studies (TWAS) of hip OA and knee OA were performed utilizing the genome-wide association study (GWAS) data of hip OA and knee OA (including 2,396 hospital-diagnosed hip OA patients versus 9,593 controls, and 4,462 hospital-diagnosed knee OA patients versus 17,885 controls) and gene expression reference to skeletal muscle and blood. The OA-associated genes identified by TWAS were further compared with the differentially expressed genes detected by the messenger RNA (mRNA) expression profiles of hip OA and knee OA. Functional enrichment and annotation analysis of identified genes was performed by the DAVID and FUMAGWAS tools.ResultsWe detected 33 common genes, eight common gene ontology (GO) terms, and one common pathway for hip OA, such as calcium and integrin-binding protein 1 (CIB1) (PTWAS = 0.025, FCmRNA = -1.575 for skeletal muscle), adrenomedullin (ADM) (PTWAS = 0.022, FCmRNA = -4.644 for blood), Golgi apparatus (PTWAS <0.001, PmRNA = 0.012 for blood), and phosphatidylinositol 3' -kinase-protein kinase B (PI3K-Akt) signalling pathway (PTWAS = 0.033, PmRNA = 0.005 for blood). For knee OA, we detected 24 common genes, eight common GO terms, and two common pathways, such as histocompatibility complex, class II, DR beta 1 (HLA-DRB1) (PTWAS = 0.040, FCmRNA = 4.062 for skeletal muscle), Follistatin-like 1 (FSTL1) (PTWAS = 0.048, FCmRNA = 3.000 for blood), cytoplasm (PTWAS < 0.001, PmRNA = 0.005 for blood), and complement and coagulation cascades (PTWAS = 0.017, PmRNA = 0.001 for skeletal muscle).ConclusionWe identified a group of OA-associated genes and pathways, providing novel clues for understanding the genetic mechanism of OA.Cite this article: Bone Joint Res. 2020;9(3):130–138.

Calcium-induced dissociation of CIB1 from ASK1 regulates agonist-induced activation of the p38 MAPK pathway in platelets.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown. Here, we show that in platelets agonist-induced activation of p38 is exclusively dependent on ASK1. Both thrombin and collagen were able to activate ASK1/p38. Activation of ASK1/p38 was strongly dependent on thromboxane A2 (TxA2) and ADP. Agonist-induced ASK1 activation is blocked by inhibition of phospholipase C (PLC) β/γ activity or by chelating intracellular Ca2+. Furthermore, treatment of platelets with thapsigargin or Ca2+ ionophore robustly induced ASK1/p38 activation. In addition, calcium and integrin-binding protein 1 (CIB1), a Ca2+-dependent negative regulator of ASK1, associates with ASK1 in resting platelets and is dissociated upon platelet activation by thrombin. Dissociation of CIB1 corresponds with ASK1 binding to tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and the autophosphorylation of ASK1 Thr838 within the catalytic domain results in full activation of ASK1. Furthermore, genetic ablation of Cib1 in mice augments agonist-induced Ask1/p38 activation. Together our results suggest that in resting platelets ASK1 is bound to CIB1 at low Ca2+ concentrations. Agonist-induced platelet activation causes an increase in intracellular Ca2+ concentration that leads to the dissociation of CIB1 from ASK1, allowing for proper dimerization through ASK1 N-terminal coiled-coil (NCC) domains.

CIB1 depletion with docetaxel or TRAIL enhances triple-negative breast cancer cell death

BackgroundPatients 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.MethodsWe 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.ResultsCIB1 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.ConclusionNovel 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.

Effects and mechanism of action of transcutaneous electrical acupuncture point stimulation in patients with abnormal semen parameters.

To evaluate the effect of transcutaneous electrical acupuncture point stimulation (TEAS) on sperm parameters and the underlying molecular mechanisms. A total of 121 patients diagnosed with oligozoospermia, asthenozoospermia or oligoasthenozoospermia were randomised into four groups (three treatment groups, one control): the TEAS groups were treated with 2 Hz (n=31), 100 Hz (n=31), or mock stimulation (n=29) at acupuncture points BL23, ST36, CV1 and CV4 for 2 months. The control group (n=30) was provided with lifestyle advice only. The changes in total sperm count and motility in the 2 Hz TEAS group were significantly greater than those in the mock group and the control group. The change in neutral α-glucosidase (NAG) and zinc levels in the 2 Hz group were significantly greater than those in the mock group and control group, and the changes in fructose levels of the 2 Hz group were significantly greater than those in the control group. Significant increases in calcium and integrin-binding protein 1 (CIB1) and reduction of cyclin-dependent kinase 1 b (CDK1) were also found after 2 Hz TEAS treatment. The present findings suggest that 2 Hz TEAS can improve sperm count and motility in patients with abnormal semen parameters, and is associated with increases in seminal plasma zinc, NAG and fructose. The upregulation of CIB1 and downregulation of CDK1 by TEAS may be associated with its positive effects on sperm motility and count. http://www.chictr.org ; registration no. ChiCTR-TRC-11001775.

Research Advances of Calcium-and Integrin-binding Protein 1 in Tumors.

Calcium-and integrin-binding protein 1(CIB1),a member of calcium binding protein containing EF-hand domain,has been shown to bind a variety of signaling proteins. Interaction of CIB1 with its various binding partners provides valuable insight into potential mechanisms by which CIB1 may regulate diverse tumors characteristic biological process that range from adhesion,migration,cell survival,proliferation,and angiogenesis. CIB1 has also been implicated in both the increase and decrease of cell proliferation.CIB1 promoted cell proliferation and survival by adjusted tumor-associated extracellular regulated protein kinases 1/2(ERK1/2),phosphatidylinositol 3 kinase(PI-3K)/v-akt murine thymoma viral oncogene homolog-dependent signaling pathway,or increased cell migration by activating Src family kinases,protein kinase C,PI-3K,and ERK 1/2-mediated signaling pathway.CIB1 inhibited cell proliferation through inhibiting polo-like kinases 3(PLK3) activity.It also inhibits cell migration by binding to and activating p21-activated kinase 1. Especially a large subset of triple-negative breast cancer may display non-oncogene addiction to CIB1,a phenomenon in which cancer cells require,or become 'addicted' to a non-mutated,non-overexpressed gene/protein that is nonetheless essential to maintain oncogenic signaling pathways,and implicate CIB1 as a novel drug target in tumors. Additionally,CIB1 plays a role in such pathological processes as cardiac hypertrophy,deafness,and male infertility. Research on CIB1 function has important clinical significance. In this review,we discussed current understanding of CIB1 in human tumors.

CIB1 contributes to oncogenic signalling by Ras via modulating the subcellular localisation of sphingosine kinase 1.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this up-regulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca2+-myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

Protein‐protein interactions between CIB1 and novel integrin binding partners: structural and thermodynamic basis of CIB1 promiscuity (979.5)

The cytoplasmic protein CIB1 (calcium‐and‐integrin‐binding protein 1) has been shown to promote cancer cell growth by affecting the RAS/RAF/MEK/ERK and the PI3K/AKT pathways, and was previously identified as an αIIb binding partner that inhibits agonist‐induced activation of the platelet‐specific integrin, αIIbβ3. To better understand the nature of the protein‐protein interactions between CIB1 and other proteins, we characterized the interaction between CIB1 and several α‐integrins. A sequence alignment of all α‐integrin CTs revealed that key residues in the CIB1 binding site on 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). A hydrophobic binding pocket on CIB1 was shown to be the major binding site for multiple α‐integrins by all‐atom replica exchange discrete molecular dynamics, competitive binding assays, and mutational analysis. We also demonstrated that CIB1 can bind to other whole integrin complexes in vivo using co‐immunopreceipitation assays. Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1‐integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function. Moreover, this work provides new insight into the nature of the protein‐protein interactions between CIB1 and its binding partners.Grant Funding Source: Supported byNHLBI 5R01HL092544 and NIGMS 5K12GM000678

CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.

Calcium- and integrin-binding protein-1 is down-regulated in the sperm of patients with oligoasthenozoospermia

The aim of this study was to determine whether altered expression and distribution of calcium- and integrin-binding protein-1 (CIB1) is involved in the pathogenesis of patients with oligoasthenozoospermia. Sperm samples were obtained from 25 infertile Chinese men who had failed to achieve conception after a period of 1-2 y and had been referred to the Reproductive Laboratory of the second hospital affiliated to the Shandong University of Traditional Chinese Medicine. Participants were divided into two groups: oligoasthenozoospermia (n = 13) and asthenozoospermia (n = 12); as a third group, fertile men (n = 19) were included as controls. The expression levels of mRNA and protein levels of CIB1 and cyclin-dependent kinase 1 (CDK1) were measured using qRT-PCR and western blotting. mRNA and protein expression levels of CIB1 were decreased in the oligoasthenozoospermia patients. Interestingly mRNA and protein expression levels of CDK1 were increased in the oligoasthenozoospermia patients. The results of the present study indicate that that CIB1 may be involved in the pathogenesis of oligoasthenozoospermia by the CDK1 signaling pathway.

Calcium- and integrin-binding protein-1 and calcineurin are upregulated in the right atrial myocardium of patients with atrial fibrillation

The aim of this study was to determine whether altered expression and distribution of calcium- and integrin-binding protein-1 (CIB1) is involved in the pathogenesis of different types of patients with atrial fibrillation (AF) associated with valvular heart disease (VHD). Right atrial specimens obtained from 65 patients undergoing valve replacement surgery were divided into three groups: sinus rhythm group (n= 24), paroxysmal atrial fibrillation group (PaAF; n= 10), and persistent atrial fibrillation group (PeAF; AF lasting >6 month; n= 31). The expression levels of mRNA and protein content for CIB1, calcineurin B, calcineurin A, and Na(+)-Ca(2+) exchanger-1 (NCX1) were measured. We also measured the combination of CIB1 with calcineurin B, L-type Ca(2+) channel, and NCX1 using immunoprecipitation. Expression of mRNA and protein content of CIB1, calcineurin B, calcineurin A, and NCX1 was increased in the AF group. Calcium- and integrin-binding protein-1 interacted with calcineurin B and L-type Ca(2+) channel. Surprisingly, CIB1 also combined with NCX1. The CIB1 and calcineurin expression was increased in AF atrial tissue and was related to the type of AF. This finding suggests that CIB1 may be involved in the pathogenesis of AF in VHD patients.

Phosphorylation Directly Regulates the Intrinsic DNA Cytidine Deaminase Activity of Activation-induced Deaminase and APOBEC3G Protein

The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.

Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca(2+)-dependent manner at the previously identified "calmodulin-binding site" of SK1. We also demonstrate that CIB1 functions as a Ca(2+)-myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling.

CIB1 and CaBP1 bind to the myo1c regulatory domain

Myo1c is a member of the myosin-I family that binds phosphoinositides and links the actin cytoskeleton to cellular membranes. Recent investigations suggest that targeting of myo1c to some subcellular regions requires the binding of an unknown protein to the IQ motifs in the myo1c regulatory domain. We identify two myristoylated proteins that bind the myo1c regulatory domain: calcium-binding protein 1 (CaBP1) and calcium- and integrin-binding-protein-1 (CIB1). CIB1 and CaBP1 interact with myo1c in vivo as determined by pull-down experiments and fluorescence microscopy where the endogenously expressed proteins show extensive cellular colocalization with myo1c. CIB1 and CaBP1 bind to the myo1c IQ motifs in the regulatory domain where they compete with calmodulin for binding. CaBP1 has a higher apparent affinity for myo1c than CIB1, and both proteins better compete with calmodulin in the presence of calcium. We propose that these proteins may play a role in specifying subcellular localization of myo1c.