Rap1 GTPase-activating Protein Research Articles

Rap1GAP exacerbates myocardial infarction by regulating the AMPK/SIRT1/NF-κB signaling pathway

Rap1 GTPase-activating protein (Rap1GAP) is an important tumor suppressor. The purpose of this study was to investigate the role of Rap1GAP in myocardial infarction (MI) and its potential mechanism. Left anterior descending coronary artery ligation was performed on cardiac-specific Rap1GAP conditional knockout (Rap1GAP-CKO) mice and control mice with MI. Seven days after MI, Rap1GAP expression in the hearts of control mice peaked, the expression of proapoptotic markers (Bax and cleaved caspase-3) increased, the expression of antiapoptotic factors (Bcl-2) decreased, and the expression of the inflammatory factors IL-6 and TNF-α increased; thus, apoptosis occurred, inflammation, infarct size, and left ventricular dysfunction increased, while the heart changes caused by MI were alleviated in Rap1GAP-CKO mice. Mouse heart tissue was obtained for transcriptome sequencing, and gene set enrichment analysis (GSEA) was used to analyze Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We found that Rap1GAP was associated with the AMPK and NF-κB signaling pathways and that Rap1GAP inhibited AMPK/SIRT1 and activated the NF-κB signaling pathway in model animals. Similar results were observed in primary rat myocardial cells subjected to oxygen–glucose deprivation (OGD) to induce ischemia and hypoxia. Activating AMPK with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) reversed the damage caused by Rap1GAP overexpression in cardiomyocytes. In addition, the coimmunoprecipitation results showed that exogenous Rap1GAP interacted with AMPK. Rap1GAP was verified to regulate the AMPK SIRT1/NF-κB signaling pathway and exacerbate the damage to myocardial cells caused by ischemia and hypoxia. In conclusion, our results suggest that Rap1GAP promotes MI by modulating the AMPK/SIRT1/NF-κB signaling pathway and that Rap1GAP may be a therapeutic target for MI treatment in the future.

Genome-wide association study reveals candidate markers related to field fertility and semen quality traits in Holstein-Friesian bulls

In vitro assessment of bull semen quality is routinely used in bull semen processing centres in order to ensure that semen destined to be used in the field has passed minimum standards. Despite these stringent quality control checks, individual bulls that pass the quality control checks can still vary in field fertility by up to 25%. A genome-wide association study was undertaken to determine genetic markers associated with prefreeze and post-thaw bull sperm quality traits as well as field fertility. Genome-wide association analysis was performed using a single nucleotide polymorphism (SNP) regression mixed linear model in WOMBAT. Genes within a 250 Kb span of a suggestive (P ≤ 1 × 10−5) SNP were considered as candidate genes. One SNP was associated with adjusted pregnancy rate, and 21 SNPs were associated across the seven semen quality traits (P ≤ 1 × 10−5). Functional candidate genes include SIPA1L2 which was associated with adjusted pregnancy rate. This encodes a Rap GTPase-activating protein involved in Rap1 signalling pathway and was previously found to play a role in the process of sperm differentiation. Gene ontology (GO) analysis also identified significantly enriched biological processes involved protein tyrosine kinase activity including genes such as DYRK1A, TEC and TXK that were associated with sperm motility prior to freezing. Another candidate gene associated with post-thaw sperm motility was FHDC1 which coordinates actin filament and microtubule dynamics. The induced 11 GO terms in the ejaculates rejected after freezing trait were related to ATPase, phosphatase and hydrolase activity. These results reveal novel specific genomic regions and candidate genes associated with economically important phenotypes such as field fertility and semen quality traits.

A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells.

The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

Mex3a promotes oncogenesis through the RAP1/MAPK signaling pathway in colorectal cancer and is inhibited by hsa-miR-6887-3p.

BackgroundAlthough Mex3 RNA‐binding family member A (Mex3a) has demonstrated an important role in multiple cancers, its role and regulatory mechanism in CRC is unclear. In this study, we aimed to investigate the role and clinical significance of Mex3a in CRC and to explore its underlying mechanism.MethodsWestern blotting and quantitative real‐time polymerase chain reaction (qRT‐PCR) were performed to detect the expression levels of genes. 5‐Ethynyl‐2'‐deoxyuridine (EDU) and transwell assays were utilized to examine CRC cell proliferation and metastatic ability. The R software was used to do hierarchical clustering analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Overexpression and rescue experiments which included U0126, a specific mitogen activated protein kinase kinase/extracellular regulated protein kinase (MEK/ERK) inhibitor, and PX‐478, a hypoxia‐inducible factor 1 subunit alpha (HIF‐1α) inhibitor, were used to study the molecular mechanisms of Mex3a in CRC cells. Co‐immunoprecipitation (Co‐IP) assay was performed to detect the interaction between two proteins. Bioinformatics analysis including available public database and Starbase software (starbase.sysu.edu.cn) were used to evaluate the expression and prognostic significance of genes. TargetScan (www.targetscan.org) and the miRDB (mirdb.org) website were used to predict the combination site between microRNA and target mRNA. BALB/c nude mice were used to study the function of Mex3a and hsa‐miR‐6887‐3p in vivo.ResultsClinicopathological and immunohistochemical (IHC) studies of 101 CRC tissues and 79 normal tissues demonstrated that Mex3a was a significant prognostic factor for overall survival (OS) in CRC patients. Mex3a knockdown substantially inhibited the migration, invasion, and proliferation of CRC cells. Transcriptome analysis and mechanism verification showed that Mex3a regulated the RAP1 GTPase activating protein (RAP1GAP)/MEK/ERK/HIF‐1α pathway. Furthermore, RAP1GAP was identified to interact with Mex3a in Co‐IP experiments. Bioinformatics and dual‐luciferase reporter experiments revealed that hsa‐miR‐6887‐3p could bind to the 3'‐untranslated regions (3'‐UTR) of the Mex3a mRNA. hsa‐miR‐6887‐3p downregulated Mex3a expression and inhibited the tumorigenesis of CRC both in vitro and in vivo.ConclusionsOur study demonstrated that the hsa‐miR‐6887‐3p/Mex3a/RAP1GAP signaling axis was a key regulator of CRC and Mex3a has the potential to be a new diagnostic marker and treatment target for CRC.

Abstract PD15-05: Sipa1 effects rock pathway in human breast cancer linking to HGF mediated changes in tight junction functions controlling metastasis

Abstract Background: SIPA1 (Signal-induced proliferation-associated gene 1) is a mitogen-inducible gene encoding a GTPase-activating protein for Rap1 and Rap2 and has been suggested to be involved in metastatic progression. We have previously shown SIPA1 may regulate barrier function in breast cancer cells as part of hepatocyte growth factor-determined changes and that this may be via a number of regulatory pathways, most likely via the ROCK pathway. Our aim with this current work was to definitively identify which pathway is involved. Methods: After knockdown of SIPA1 in MDA-MB-231 cells, they were treated to a number of inhibitors to proteins involved in pathways involved in TJ barrier function (ROCK, N-WASP, ARPs, WAVEs, MAPK, ERK, PLC-gamma). Following the identification of suitable inhibitor a large scale in vivo study was carried out using a tumour model of SIPA1 knockdown versus WT control was treated with/without inhibitors and immunohistochemistry (IHC) used to assess changes in tumour growth, architecture and protein expression. We also assessed the expression of SIPA-1 in a cohort of human breast cancer tissues using IHC. Results: After knockdown of SIPA1 in MDA-MB-231 cells, we subjected cells to a number of inhibitors to proteins involved in pathways involved barrier function (ROCK, N-WASP, ARPs, WAVEs, MAPK, ERK, PLC-gamma). Analysis of behaviour showed that knockdown cells no longer responded to the ROCK inhibitor (ROCKi Y-27632, p<0.05, n=10). In vivo tumour samples were assessed for expression of ROCKI, expression of phospho-ROCK, and any differences when treated with ROCKi. Control tissues showed good levels of all three proteins (SIPA1, ROCKI and phosphor-ROCK). Knockdown tissues exhibited significantly lower levels of ROCK and phosphor-ROCK (p<0.01, n=12). In control tumours, phosphor-ROCK was increased after treatment with ROCKi; conversely, in SIPA1 knockdown tumours, phosphor-ROCK was decreased after treatment with ROCKi (p<0.05). In SIPA1 knockdown tissues, ROCK and phosphor-ROCK was diffuse in staining pattern when compared to the control where the staining was located at the cell membrane. Conclusion: Our results confirm our hypothesis that SIPA1 is involved in the control of TJ in breast cancer and that this is through the ROCK pathway. Moreover, these results demonstrate that this may be due to the effect that SIPA1 has on cellular ratios of ROCK to phosphor-ROCK in breast cancer. Citation Format: Chang Liu, Fiona Ruge, Lin Ye, Gregory M. Harrison, Wen G. Jiang, Tracey A. Martin. Sipa1 effects rock pathway in human breast cancer linking to HGF mediated changes in tight junction functions controlling metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-05.

Multi-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability

SYNGAP1 is a neuronal Ras and Rap GTPase-activating protein with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia, and autism spectrum disorder (ASD), whether and how they contribute to individual disease phenotypes is often unknown. Here, we characterize 57 variants in seven assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure variant impact on protein stability, pERK, pGSK3β, pp38, pCREB, and high-content imaging to examine subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wild-type (WT)-like in their regulation of pERK and pGSK3β, while all variants retain at least partial ability to dephosphorylate pCREB. Interestingly, our assays reveal that a larger proportion of variants located within the disordered domain of unknown function (DUF) comprising the C-terminal half of SYNGAP1 exhibited higher LoF, compared to variants within the better studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction for only two missense variants, both located within the catalytic domain. Using high-content imaging, we find variants located within the C2 domain known to mediate membrane lipid interactions exhibit significantly larger cytoplasmic speckles than WT SYNGAP1. Moreover, this subcellular phenotype shows both correlation with altered catalytic activity and unique deviation from signaling assay results, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional datasetallows clustering of variants based on functional phenotypes and provides high-confidence, multi-functional measures for making pathogenicity predictions.

SIPA1L2 controls trafficking and local signaling of TrkB-containing amphisomes at presynaptic terminals

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.

Rap1 signal modulators control the maintenance of hematopoietic progenitors in bone marrow and adult long-term hematopoiesis.

Adult long‐term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self‐renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase‐activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane‐targeted C3G (C3G‐F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long‐term hematopoiesis of all cell lineages. C3G‐F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G‐F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G‐F expression caused a markedly prolonged activation of c‐Kit receptor and downstream signaling through SCF ligation. A minor population of C3G‐F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long‐term adult hematopoiesis.

Phosphorylation of synaptic GTPase-activating protein (synGAP) by polo-like kinase (Plk2) alters the ratio of its GAP activity toward HRas, Rap1 and Rap2 GTPases

SynGAP is a Ras and Rap GTPase-activating protein (GAP) found in high concentration in the postsynaptic density (PSD) fraction from mammalian forebrain where it binds to PDZ domains of PSD-95. Phosphorylation of pure recombinant synGAP by Ca2+/calmodulin-dependent protein kinase II (CaMKII) shifts the balance of synGAP's GAP activity toward inactivation of Rap1; whereas phosphorylation by cyclin-dependent kinase 5 (CDK5) has the opposite effect, shifting the balance toward inactivation of HRas. These shifts in balance contribute to regulation of the numbers of surface AMPA receptors, which rise during synaptic potentiation (CaMKII) and fall during synaptic scaling (CDK5). Polo-like kinase 2 (Plk2/SNK), like CDK5, contributes to synaptic scaling. These two kinases act in concert to reduce the number of surface AMPA receptors following elevated neuronal activity by tagging spine-associated RapGAP protein (SPAR) for degradation, thus raising the level of activated Rap. Here we show that Plk2 also phosphorylates and regulates synGAP. Phosphorylation of synGAP by Plk2 stimulates its GAP activity toward HRas by 65%, and toward Rap1 by 16%. Simultaneous phosphorylation of synGAP by Plk2 and CDK5 at distinct sites produces an additive increase in GAP activity toward HRas (∼230%) and a smaller, non-additive increase in activity toward Rap1 (∼15%). Dual phosphorylation also produces an increase in GAP activity toward Rap2 (∼40–50%), an effect not produced by either kinase alone. As we previously observed for CDK5, addition of Ca2+/CaM causes a substrate-directed doubling of the rate and stoichiometry of phosphorylation of synGAP by Plk2, targeting residues also phosphorylated by CaMKII. In summary, phosphorylation by Plk2, like CDK5, shifts the ratio of GAP activity of synGAP to produce a greater decrease in active Ras than in active Rap, which would produce a shift toward a decrease in the number of surface AMPA receptors in neuronal dendrites.

The downregulation of Rap1 GTPase-activating protein is associated with a poor prognosis in colorectal cancer and may impact on tumor progression.

Rap1 GTPase-activating protein (Rap1GAP) has been reported to serve an important role in various types of cancer by specific stimulation as a negative regulator of Rap1 activity. However, the role of Rap1GAP in colorectal cancer (CRC) has yet to be fully elucidated. The aim of the present study was to investigate the expression of Rap1GAP in CRC tissues and to elucidate its clinical significance. The expression of Rap1GAP, matrix metallopeptidase 9 (MMP-9) and E-cadherin in 227 CRC tissues and paired para-carcinoma tissues was detected by immunohistochemistry. Associations between Rap1GAP expression and clinicopathological characteristics, and between Rap1GAP expression and prognostic value (OS + DFS) in CRC were investigated. Furthermore, associations between Rap1GAP expression and MMP-9 expression, and between Rap1GAP expression and E-cadherin expression were also investigated. Rap1GAP expression was markedly downregulated in CRC tissues compared with para-carcinoma tissues. Decreased expression of Rap1GAP was significantly associated with depth of invasion, lymph node metastasis, advanced Tumor-Node-Metastasis stage and a poor prognosis in patients with CRC following surgery. Furthermore, univariate and multivariate analyses revealed that Rap1GAP was an independent poor prognostic factor for disease-free survival and overall survival. In addition, Rap1GAP expression was negatively associated with MMP-9 and positively associated with E-cadherin in 227 CRC samples. In brief, the results of the present study suggested that Rap1GAP may be involved in tumor progression in CRC and may serve as a potential target for prognostic prediction of patients with CRC.

Sipa1 deficiency–induced bone marrow niche alterations lead to the initiation of myeloproliferative neoplasm

AbstractMutations of signal-induced proliferation-associated gene 1 (SIPA1), a RAP1 GTPase-activating protein, were reported in patients with juvenile myelomonocytic leukemia, a childhood myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Sipa1 deficiency in mice leads to the development of age-dependent MPN. However, Sipa1 expression in bone marrow (BM) microenvironment and its effect on the pathogenesis of MPN remain unclear. We here report that Sipa1 is expressed in human and mouse BM stromal cells and downregulated in these cells from patients with MPN or MDS/MPN at diagnosis. By using the Sipa1−/− MPN mouse model, we find that Sipa1 deletion causes phenotypic and functional alterations of BM mesenchymal stem and progenitor cells prior to the initiation of the MPN. Importantly, the altered Sipa1−/− BM niche is required for the development of MDS/MPN following transplantation of normal hematopoietic cells. RNA sequencing reveals an enhanced inflammatory cytokine signaling and dysregulated Dicer1, Kitl, Angptl1, Cxcl12, and Thpo in the Sipa1−/− BM cellular niches. Our data suggest that Sipa1 expression in the BM niche is critical for maintaining BM niche homeostasis. Moreover, Sipa1 loss–induced BM niche alterations likely enable evolution of clonal hematopoiesis to the hematological malignancies. Therefore, restoring Sipa1 expression or modulating the altered signaling pathways involved might offer therapeutic potential for MPN.

Sipa1 deficiency unleashes a host-immune mechanism eradicating chronic myelogenous leukemia-initiating cells

Chronic myelogenous leukemia (CML) caused by hematopoietic stem cells expressing the Bcr-Abl fusion gene may be controlled by Bcr-Abl tyrosine kinase inhibitors (TKIs). However, CML-initiating cells are resistant to TKIs and may persist as minimal residual disease. We demonstrate that mice deficient in Sipa1, which encodes Rap1 GTPase-activating protein, rarely develop CML upon transfer of primary hematopoietic progenitor cells (HPCs) expressing Bcr-Abl, which cause lethal CML disease in wild-type mice. Resistance requires both T cells and nonhematopoietic cells. Sipa1−/− mesenchymal stroma cells (MSCs) show enhanced activation and directed migration to Bcr-Abl+ cells in tumor tissue and preferentially produce Cxcl9, which in turn recruits Sipa1−/− memory T cells that have markedly augmented chemotactic activity. Thus, Sipa1 deficiency uncovers a host immune mechanism potentially capable of eradicating Bcr-Abl+ HPCs via coordinated interplay between MSCs and immune T cells, which may provide a clue for radical control of human CML.

Relationship between methylation status of Rap1 GTPase activating protein and colon cancer

Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People's Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43% (50.35%), 21.37% (8.39%) and 17.43% (15.71%) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P 60 years old: 36.26% (62.62%) and 26.23% (76.42%); well-differentiated vs. moderately/poorly-differentiated: 21.98% (40.32%) vs. 42.74% (74.20%); TNM Ⅰ-Ⅱ vs Ⅲ-Ⅳ: 25.31% (48.27%) vs. 36.26% (75.55%); all P > 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer. Key words: Colon neoplasms; RAP1 GTPase activating protein; Methylation; Quantitative methylation specific polymerase chain reaction

In Vitro Assay for the Rap GTPase-Activating Protein Activity of the Purified Cytoplasmic Domain of Plexin.

Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.

Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.)

The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H+-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron–sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted.

Sipa1l3/SPAR3 is targeted to postsynaptic specializations and interacts with the Fezzin ProSAPiP1/Lzts3.

Rap GTPase-activating proteins (RapGAPs) are essential for synaptic function as they tightly regulate synaptic Rap signaling. Among the most abundant synaptic RapGAPs in brain are the Spine-associated RapGAPs (SPARs) Sipa1l1/SPAR and Sipa1l2/SPAR2, whereas nothing has been reported on Sipa1l3/SPAR3. In this study, we show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in the developing rat brain and is localized at excitatory postsynapses. We further demonstrate that the Sipa1l3/SPAR3 C-terminus is required for postsynaptic targeting and represents an interaction module for Fezzins such as ProSAPiP1/Lzts3, a binding partner of the postsynaptic scaffold protein Shank3. Taken together, our data imply that Sipa1l3/SPAR3 is a hitherto unknown synaptic RapGAP, which is targeted to postsynaptic specializations and interacts with Fezzins. Spine-associated RapGAPs (SPARs) are essential modulators of synaptic signaling. Our study is the first to characterize the SPAR family member Sipa1l3/SPAR3 in neuronal tissue. We show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in brain and is localized to excitatory postsynapses via its C-terminus, which represents an interaction module for other postsynaptic proteins including the Fezzin ProSAPiP1/Lzts3.

Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4) activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM), as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM) cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.

Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.

High SIPA-1 expression in proximal tubules of human kidneys under pathological conditions.

Systemic lupus erythematosus (SLE) and clear cell renal cell carcinoma (CC-RCC) are serious disorders and usually fatal, and always accompanied with pathological changes in the kidney. Signal-induced proliferation-associated protein 1 (SIPA-1) is a Rap1GTPase activating protein (Rap1GAP) expressed in the normal distal and collecting tubules of the murine kidney. Lupus-like autoimmune disease and leukemia have been observed in SIPA-1 deficient mice, suggesting a pathological relevance of SIPA-1 to SLE and carcinoma in human being. The expression pattern of SIPA-1 is as yet undefined and the pathogenesis of these diseases in humans remains elusive. In this study, we used both immunohistochemistry and quantum dot (QD)-based immunofluorescence staining to investigate the expression of SIPA-1 in renal specimens from SLE and CC-RCC patients. MTT assay and Western blotting were employed to evaluate the effects of SIPA-1 overexpression on the proliferation and apoptosis of renal cell lines. Semi-quantitative reverse transcriptase-PCR (RT-PCR) was applied to examine the changes of hypoxia-inducible factor-1α (HIF-1α) mRNA level. Results showed that SIPA-1 was highly expressed in the proximal and collecting tubules of nephrons in SLE patients compared to normal ones, and similar results were obtained in the specimens of CC-RCC patients. Although SIPA-1 overexpression did not affect cellular proliferation and apoptosis of both human 786-O renal cell carcinoma cells and rat NRK-52E renal epithelial cell lines, RT-PCR results showed that HIF-1α mRNA level was down-regulated by SIPA-1 overexpression in 786-O cells. These findings suggest that SIPA-1 may play critical roles in the pathological changes in kidney, and might provide a new biomarker to aid in the diagnosis of SLE and CC-RCC.

The small GTPase Rap1 promotes cell movement rather than stabilizes adhesion in epithelial cells responding to insulin-like growth factor I.

The Ras-related GTPase Rap1 promotes cell adhesion and migration. Although the significance of Rap1 contribution to cell migration is increasingly being recognized, little is known about the biochemical mechanisms driving this process. In the present study, we discovered a previously unidentified regulatory role of insulin-like growth factor typeI (IGF-I) receptor (IGF-IR) in CRK Src homology 3 (SH3)-binding guanine-nucleotide-releasing protein (C3G)-Rap1-fascin-actin axis promoting cell movement. We demonstrate that a burst of Rap1 activity, rather than presumed hyperactivation, is imperative for the onset of cell movement. We show that while autophosphorylated IGF-IR signals to C3G to activate Rap1, subsequent IGF-IR internalization promotes gradual inactivation of Rap1 by putative Rap1 GTPase-activating protein (GAP). Additionally, IGF-IR signalling recruits active Rap1 at sites of cell motile protrusions. C3G depletion prevents IGF-I-induced fascin accumulation at actin microspikes and blocks protrusions. In the absence of IGF-IR activity, the wild-type (WT) Rap1 and the constitutively active V12Rap1 mutant remain in cell-cell contacts. Forced inactivation of Rap1 signalling by overexpressing dominant negative N17Rap1, Rap1GAP or by silencing C3G has a detrimental effect on filamentous (F)-actin and cell adhesion irrespective of IGF-IR signalling. We conclude that the basal levels of Rap1 activity holds up cell adhesion, whereas sequential regulation of C3G and GAP by IGF-IR reverses the labile Rap1 function from supporting adhesion to promoting migration.