MgcRacGAP, a Rho GAP essential to cytokinesis, works both as a Rho GTPase regulator and as a scaffolding protein. MgcRacGAP interacts with MKLP1 to form the centralspindlin complex and associates ...with the RhoGEF Ect2. The GAP activity of MgcRacGAP is regulated by Aurora B phosphorylation. We have isolated B56ε, a PP2A regulatory subunit, as a new MgcRacGAP partner. We report here that (i) MgcRacGAP is phosphorylated by Aurora B and Cdk1, (ii) PP2A dephosphorylates Aurora B and Cdk1 phosphorylated sites and (iii) inhibition of PP2A abrogates MgcRacGAP/Ect2 interaction. Therefore, PP2A may regulate cytokinesis by dephosphorylating MgcRacGAP and its interacting partners.
Structured summary:MINT-6166761:Aurora B (uniprotkb:Q96GD4) phosphorylates (MI:0217) MgcRacGAP (uniprotkb:Q9H0H5) by protein kinase assay (MI:0424)MINT-6166774:Cdk1 (uniprotkb:P06493) phosphorylates (MI:0217) MgcRacGAP (uniprotkb:Q9H0H5) by protein kinase assay (MI:0424)MINT-6166653:PP2A (uniprotkb:P67775) dephosphorylates (MI:0203) MgcRacGAP (uniprotkb:Q9H0H5) by phosphatase assay (MI:0434)MINT-6166710, MINT-6166727, MINT-6166735:MgcRacGAP (uniprotkb:Q9H0H5)physically interacts (MI:0218) with B56ε (uniprotkb:Q16537) by coimmunoprecipitation (MI:0019)MINT-6166691:B56ε (uniprotkb:Q16537) physically interacts (MI:0218) with MgcRacGAP (uniprotkb:Q9H0H5) by far western blotting (MI:0047)MINT-6166556:MgcRacGAP (uniprotkb:Q9H0H5) physically interacts (MI:0218) with B56ε (uniprotkb:Q16537) by two-hybrid (MI:0018)MINT-6166634:MgcRacGAP (uniprotkb:Q9H0H5) physically interacts (MI:0218) with Ect2 (uniprotkb:Q9H8V3) by coimmunoprecipitation (MI:0019)MINT-6166596:MKLP-1 (uniprotkb:Q02241) physically interacts (MI:0218) with B56ε (uniprotkb:Q16537) , MgcRacGAP (uniprotkb:Q9H0H5), PP2A (uniprotkb:P67775) bycoimmunoprecipitation (MI:0019)
Oculocerebrorenal Lowe syndrome is a rare X-linked disorder characterized by bilateral cataract, mental retardation and renal Fanconi syndrome. The Lowe syndrome protein Ocrl1 is a PIP2 ...5-phosphatase, primarily localized to the trans-Golgi network (TGN), which ‘loss of function’ mutations result in PIP2 accumulation in patient's cells. Although PIP2 is involved in many cell functions including signalling, vesicle trafficking and actin polymerization, it has been difficult so far to decipher molecular/cellular mechanisms responsible for Lowe syndrome phenotype. We have recently shown that, through its C-terminal RhoGAP domain, Ocrl1 forms a stable complex with Rac GTPase within the cell. In line with this finding, we report here that upon epidermal growth factor induced Rac activation in COS-7 cells, a fraction of Ocrl1 translocates from TGN to plasma membrane and concentrates in membrane ruffles. In order to investigate the functionality of Ocrl1 in plasma membrane, we have analysed PIP2 distribution in human dermal fibroblasts (HDFs) from Lowe patients versus control HDFs. As revealed by both immunodetection and green fluorescent protein–PH binding, PIP2 was found strikingly to accumulate in PDGF induced ruffles in Lowe HDFs when compared with control. This suggests that Ocrl1 is active as a PIP2 5-phosphatase in Rac induced membrane ruffles. Cellular properties such as cell migration and establishment of cell–cell contacts, which depend on ruffling and lamellipodia formation, should be further investigated to understand the pathophysiology of Lowe syndrome.
RhoGTPases (Rho, Rac, and Cdc42) are known to regulate multiple functions, including cell motility, adhesion, and proliferation; however, the signaling pathways underlying these pleiotropic effects ...are far from fully understood. We have recently described a new RhoGAP (GTPase activatingprotein for RhoGTPases) gene, MgcRacGAP, primarily expressed in male germ cells, at the spermatocyte stage. We report here the isolation, through two-hybrid cloning, of a new partner of MgcRacGAP, very specifically expressed in the male germ line and showing structural features of anion transporters. This large protein (970 amino acids and a predicted size of 109 kDa), we provisionally designated Tat1 (for testis anion transporter 1), is closely related to a sulfate permease family comprising three proteins in human (DRA, Pendrin, and DTD); it is predicted to be an integral membrane protein with 14 transmembrane helices and intracytoplasmic NH2 and COOH termini. In situ hybridization studies demonstrate that Tat1 and MgcRacGAPgenes are coexpressed in male germ cells at the spermatocyte stage. On testis sections, Tat1 protein can be immunodetected in spermatocytes and spermatids associated with plasma membrane. Two-hybrid and in vitro binding assays demonstrate that MgcRacGAP stably interacts through its NH2-terminal domain with the Tat1 COOH-terminal region. Expression of Tat1 protein in COS7 cells generates a 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene and chloride-sensitive sulfate transport. Therefore we conclude that Tat1 is a novel sulfate transporter specifically expressed in spermatocytes and spermatids and interacts with MgcRacGAP in these cells. These observations raise the possibility of a new regulatory pathway linking sulfate transport to Rho signaling in male germ cells.
In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure
a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family ...GTPases. A specific mRNA of 3.2 kilobases was detected
in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we
call MgcRacGAP, was detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific
to recombinant protein. In vitro , the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal
to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs.
The closest homolog of MgcRacGAP is RotundRacGAP, a product of the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support
the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility.
NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation
of this multimolecular enzyme minimally requires assembly at the ...membrane with flavocytochrome b of cytosolic components p47 , p67 , and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated
with NADPH oxidase activation in vitro ; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating
in vivo interactions between human p47 , p67 , and Rac proteins. Rac proteins interact with p67 in a GTP-dependent manner, but do not interact with p47 . Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67 ; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67 . Finally, we observe that p67 interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47 and p67 . These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67 and that Rac2 is the main p67 -interacting GTPase in human cells.
Philadelphia (Ph)-positive leukemias invariably contain a chromosomal translocation fusing
BCR to
ABL. The BCR-ABL protein is responsible for leukemogenesis. Here we show that exposure of
bcr-null ...mutant mice to gram-negative endotoxin led to severe septic shock and increased tissue injury by neutrophils. Neutrophils of
bcr (-/-) mice showed a pronounced increase in reactive oxygen metabolite production upon activation and were more sensitive to priming stimuli. Activated (-/-) neutrophils displayed a 3-fold increased p21
rac2
membrane translocation compared with (+/+) neutrophils. These results connect Bcr in vivo with the regulation of Rac-mediated superoxide production by the NADPH-oxidase system of leukocytes and suggest a link between Bcr function and the cell type affected in Ph-positive leukemia.
Oligophrenin-1 regulates dendritic spine morphology in the brain. Mutations in the oligophrenin-1 gene (OPHN1) cause intellectual disability. We discovered a previously unknown partner of ...oligophrenin-1, Rev-erbα, a nuclear receptor that represses the transcription of circadian oscillators. We found that oligophrenin-1 interacts with Rev-erbα in the mouse brain, causing it to locate to dendrites, reducing its repressor activity and protecting it from degradation. Our results indicate the presence of a circadian oscillator in the hippocampus, involving the clock gene Bmal1 (also known as Arntl), that is modulated by Rev-erbα and requires oligophrenin-1 for normal oscillation. We also found that synaptic activity induced Rev-erbα localization to dendrites and spines, a process that is mediated by AMPA receptor activation and requires oligophrenin-1. Our data reveal new interactions between synaptic activity and circadian oscillators, and delineate a new means of communication between nucleus and synapse that may provide insight into normal plasticity and the etiology of intellectual disability.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
OCRL mutations are associated with both Lowe syndrome and Dent-2 disease, two rare X-linked conditions. Lowe syndrome is an oculo-cerebro-renal disorder, whereas Dent-2 patients mainly present renal ...proximal tubulopathy. Loss of OCRL-1, a phosphoinositide-5-phosphatase, leads in Lowe patients' fibroblasts to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) accumulation, with defects in F-actin network, α-actinin distribution and ciliogenesis, whereas fibroblasts of Dent-2 patients are still uncharacterized. To search for mechanisms linked to clinical variability observed between these two OCRL mutation-associated pathologies, we compared dermal fibroblasts from independent patients, four affected by Dent-2 disease and six with Lowe syndrome. For the first time, we describe that Dent-2 fibroblasts with OCRL loss-of-function (LOF) mutations exhibit decrease in actin stress fibers, appearance of punctate α-actinin signals and alteration in primary cilia formation. Interestingly, we quantified these phenotypes as clearly intermediate between Lowe and control fibroblasts, thus suggesting that levels of these defects correlate with clinical variations observed between patients with OCRL mutations. In addition, we show that Lowe and Dent-2 fibroblasts display similar PI(4,5)P2 accumulation levels. Finally, we analyzed INPP5B, a paralogous gene already reported to exhibit functional redundancy with OCRL, and report neither differences in its expression at RNA or protein levels, nor specific allelic variations between fibroblasts of patients. Altogether, we describe here differential phenotypes between fibroblasts from Lowe and Dent-2 patients, both associated with OCRL LOF mutations, we exclude direct roles of PI(4,5)P2 and INPP5B in this phenotypic variability and we underline potential key alterations leading to ocular and neurological clinical features in Lowe syndrome.
ABSTRACT
Dent‐2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5‐phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic ...kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo‐exon inclusion in the mRNA, leading to a OCRL‐1 protein loss. An exon‐skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7‐modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL‐1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL‐1 protein, even at a reduced level, through RNA‐based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.
Manipulation of mRNA composition can represent a personalized therapeutic approach to treat splice mutations. We demonstrate here the restoration of a functional OCRL‐1 protein and the correction of pathological phenotypes on cells from a patient with oculocerebrorenal Lowe syndrome treated with an antisens oligonucleotide (AON) blocking the effect of the mutation.
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