Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU.

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

Inactivation of fission yeast Erh1 de-represses pho1 expression: evidence that Erh1 is a negative regulator of prt lncRNA termination.

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1, which is normally repressed by transcription in cis of a 5' flanking prt lncRNA. Here we present evidence that de-repression of pho1 by erh1Δ is achieved through precocious 3'-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1Δ depends on CPF (cleavage and polyadenylation factor) subunits Ctf1, Dis2, Ssu72, Swd22, and Ppn1 and on termination factor Rhn1; (iii) de-repression requires synthesis by the Asp1 IPP kinase of inositol 1-pyrophosphates (1-IPPs); (iv) de-repression is effaced by mutating Thr4 of the RNA polymerase II CTD to alanine; and (v) erh1Δ exerts an additive effect on pho1 de-repression in combination with mutating CTD Ser7 to alanine and with deletion of the IPP pyrophosphatase Aps1. These findings point to Erh1 as an antagonist of lncRNA termination in the prt-pho1 axis. In contrast, in mmi1Δ cells there is a reduction in pho1 mRNA and increase in the formation of a prt-pho1 read-through transcript, consistent with Mmi1 being an agonist of prt termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during prt lncRNA synthesis. Consistent with this idea, erh1Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA.

Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis.

Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

A two-way switch for inositol pyrophosphate signaling: Evolutionary history and biological significance of a unique, bifunctional kinase/phosphatase.

The inositol pyrophosphates (PP-InsPs) are a unique subgroup of intracellular signals with diverse functions, many of which can be viewed as reflecting an overarching role in metabolic homeostasis. Thus, considerable attention is paid to the enzymes that synthesize and metabolize the PP-InsPs. One of these enzyme families - the diphosphoinositol pentakisphosphate kinases (PPIP5Ks) - provides an extremely rare example of separate kinase and phosphatase activities being present within the same protein. Herein, we review the current state of structure/function insight into the PPIP5Ks, the separate specialized activities of the two metazoan PPIP5K genes, and we describe a phylogenetic analysis that places PPIP5K evolutionary origin within the Excavata, the very earliest of eukaryotes. These different aspects of PPIP5K biology are placed in the context of a single, overriding question. Why are they bifunctional: i.e., what is the particular significance of the ability to turn PP-InsP signaling on or off from two separate 'switches' in a single protein?

Inositol phosphate kinases: Expanding the biological significance of the universal core of the protein kinase fold.

The protein kinase family is characterized by substantial conservation of architectural elements that are required for both ATP binding and phosphotransferase activity. Many of these structural features have also been identified in homologous enzymes that phosphorylate a variety of alternative, non-protein substrates. A comparative structural analysis of these different kinase sub-classes is a portal to a greater understanding of reaction mechanisms, enzyme regulation, inhibitor-development strategies, and superfamily-level evolutionary relationships. To serve such advances, we review structural elements of the protein kinase fold that are conserved in the subfamily of inositol phosphate kinases (InsPKs) that share a PxxxDxKxG catalytic signature: inositol 1,4,5-trisphosphate kinase (IP3K), inositol hexakisphosphate kinase (IP6K), and inositol polyphosphate multikinase (IPMK). We describe conservation of the fundamental two-lobe kinase architecture: an N-lobe constructed upon an anti-parallel ß-strand scaffold, which is coupled to a largely helical C-lobe by a single, adenine-binding hinge. This equivalency also includes a G-loop that embraces the ß/γ-phosphates of ATP, a transition-state stabilizing residue (Lys/His), and a Mg-positioning aspartate residue within a catalytic triad. Furthermore, we expand this list of conserved structural features to include some not previously identified in InsPKs: a 'gatekeeper' residue in the N-lobe, and an 'αF'-like helix in the C-lobe that anchors two structurally-stabilizing, hydrophobic spines, formed from non-consecutive residues that span the two lobes. We describe how this wide-ranging structural homology can be exploited to develop lead inhibitors of IP6K and IPMK, by using strategies similar to those that have generated ATP-competing inhibitors of protein-kinases. We provide several examples to illustrate how such an approach could benefit human health.

Crystal Structure and Product Analysis of an Archaeal myo-Inositol Kinase Reveal Substrate Recognition Mode and 3-OH Phosphorylation.

The TK2285 protein from Thermococcus kodakarensis was recently characterized as an enzyme catalyzing the phosphorylation of myo-inositol. Only two myo-inositol kinases have been identified so far, the TK2285 protein and Lpa3 from Zea mays, both of which belong to the ribokinase family. In either case, which of the six hydroxyl groups of myo-inositol is phosphorylated is still unknown. In addition, little is known about the myo-inositol binding mechanism of these enzymes. In this work, we determined two crystal structures: those of the TK2285 protein complexed with the substrates (ATP analogue and myo-inositol) or the reaction products formed by the enzyme. Analysis of the ternary substrates-complex structure and site-directed mutagenesis showed that five residues were involved in the interaction with myo-inositol. Structural comparison with other ribokinase family enzymes indicated that two of the five residues, Q136 and R140, are characteristic of myo-inositol kinase. The crystal structure of the ternary products-complex, which was prepared by incubating the TK2285 protein with myo-inositol and ATP, holds 1d-myo-inositol 3-phosphate (Ins(3)P) in the active site. NMR and HPLC analyses with a chiral column also indicated that the TK2285 reaction product was Ins(3)P. The results obtained here showed that the TK2285 protein specifically catalyzes the phosphorylation of the 3-OH of myo-inositol. We thus designated TK2285 as myo-inositol 3-kinase (MI3K). The precise identification of the reaction product should provide a sound basis to further explore inositol metabolism in Archaea.

PI3P binding by Atg21 organises Atg8 lipidation.

Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 ß-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.

Comparison of Saccharomyces cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site.

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.

Identification of differentially coexpressed genes in gonadotrope tumors and normal pituitary using bioinformatics methods.

To investigate the underlying molecular mechanisms of pituitary tumor by using the microarray expression profiles of pituitary tumor and normal tissue samples. The gene expression profile of GSE26966 was downloaded from Gene Expression Omnibus, including nine normal samples and 14 pituitary tumor samples. The differentially coexpressed genes (DEGs) were identified by Affy package in R Software. The functional and pathway enrichment analysis of the screened DEGs were performed by DAVID. Then, differential coexpression networks were contructed and further analyzed. Functional and pathway enrichment analysis of the 1220 identified DEGs revealed that phosphatidylinositol signaling system, p53 signaling pathway and inositol phosphate metabolism were disturbed in pituitary tumors. The degree of DLK1, CDKN2A and ITGA4 in the constructed differential coexpression network was 46, 45 and 44, respectively. In addition, MPP2 and ASAP2 were the obvious hub genes in the constructed differential coexpression network. Through exploring genes in the differential coexpression networks, the results suggested that DLK1, CDKN2A, ITGA4, MPP2 and ASAP2 may potentially be used as biomarkers for pituitary tumor.

GmSAL1 hydrolyzes inositol-1,4,5-trisphosphate and regulates stomatal closure in detached leaves and ion compartmentalization in plant cells.

Inositol polyphosphatases are important regulators since they control the catabolism of phosphoinositol derivatives, which are often signaling molecules for cellular processes. Here we report on the characterization of one of their members in soybean, GmSAL1. In contrast to the substrate specificity of its Arabidopsis homologues (AtSAL1 and AtSAL2), GmSAL1 only hydrolyzes inositol-1,4,5-trisphosphate (IP3) but not inositol-1,3,4-trisphosphate or inositol-1,4-bisphosphate.The ectopic expression of GmSAL1 in transgenic Arabidopsis thaliana led to a reduction in IP3 signals, which was inferred from the reduction in the cytoplasmic signals of the in vivo biomarker pleckstrin homology domain-green florescent protein fusion protein and the suppression of abscisic acid-induced stomatal closure. At the cellular level, the ectopic expression of GmSAL1 in transgenic BY-2 cells enhanced vacuolar Na(+) compartmentalization and therefore could partially alleviate salinity stress.

Regulation of inositol metabolism is fine-tuned by inositol pyrophosphates in Saccharomyces cerevisiae.

Although inositol pyrophosphates have diverse roles in phosphate signaling and other important cellular processes, little is known about their functions in the biosynthesis of inositol and phospholipids. Here, we show that KCS1, which encodes an inositol pyrophosphate kinase, is a regulator of inositol metabolism. Deletion of KCS1, which blocks synthesis of inositol pyrophosphates on the 5-hydroxyl of the inositol ring, causes inositol auxotrophy and decreased intracellular inositol and phosphatidylinositol. These defects are caused by a profound decrease in transcription of INO1, which encodes myo-inositol-3-phosphate synthase. Expression of genes that function in glycolysis, transcription, and protein processing is not affected in kcs1Δ. Deletion of OPI1, the INO1 transcription repressor, does not fully rescue INO1 expression in kcs1Δ. Both the inositol pyrophosphate kinase and the basic leucine zipper domains of KCS1 are required for INO1 expression. Kcs1 is regulated in response to inositol, as Kcs1 protein levels are increased in response to inositol depletion. The Kcs1-catalyzed production of inositol pyrophosphates from inositol pentakisphosphate but not inositol hexakisphosphate is indispensable for optimal INO1 transcription. We conclude that INO1 transcription is fine-tuned by the synthesis of inositol pyrophosphates, and we propose a model in which modulation of Kcs1 controls INO1 transcription by regulating synthesis of inositol pyrophosphates.

The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinase Cδ cascade.

Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP4) and InsP5, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

[Cloning, expression and immune function analysis of a gene encoding inositol monophosphate in Schistosoma japonicum].

OBJECTIVE: To clone and express a full-length cDNA encoding inositol monophosphate of Schistosoma japonicum (SjIM), and to access its immunoprotection in BALB/c mice for schistosomisis. METHODS: A full-length cDNA encoding the S. japonicum inositol monophosphate was isolated from 42 d schistosomes cDNAs. The expression profiles in different developmental stages were detected by real-time quantitative RT-PCR. The open reading frame (ORF) was subcloned into a pET28a(+) vector and transformed into BL21 and the recombinant protein was induced by IPTG. The immune characters of the purified recombinant protein were analyzed by Western blotting and immunoprotection in BALB/c mice. RESULTS: Bioinformatics analysis indicated that SjIM had an ORF of 834 base pairs that encoded 278 amino acids. Real-time quantitative RT-PCR analysis revealed that SjIM was upregulated in 35-day-old schistosomes, while the expression level in females was higher than that in male worms in 42nd day. Western blotting showed that the recombinant SjIM was immunogenic. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjIM could induce 48.76% and 41.29% reductions in the numbers of worms and eggs in the liver, respectively. CONCLUSIONS: The gene of SjIM is obtained from schistosomes cDNAs and the recombinant SjIM protein is induced successfully in E. coli. These aforementioned results demonstrate that the recombinant SjIM cand induce partial protection against schistosomiasis in BALB/c mice.

Cloning, expression, purification, crystallization and X-ray analysis of inositol monophosphatase from Mus musculus and Homo sapiens.

Inositol monophosphatase (IMPase) catalyses the hydrolysis of inositol monophosphate to inositol and is crucial in the phosphatidylinositol (PI) signalling pathway. Lithium, which is the drug of choice for bipolar disorder, inhibits IMPase at therapeutically relevant plasma concentrations. Both mouse IMPase 1 (MmIMPase 1) and human IMPase 1 (HsIMPase 1) were cloned into pRSET5a, expressed in Escherichia coli, purified and crystallized using the sitting-drop method. The structures were solved at resolutions of 2.4 and 1.7 Å, respectively. Comparison of MmIMPase 1 and HsIMPase 1 revealed a core r.m.s. deviation of 0.516 Å.

Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs. parasitic Apicomplexa (Plasmodium, Toxoplasma).

The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also "Conclusions" section.

Prolyl oligopeptidase, inositol phosphate signalling and lithium sensitivity.

Inhibition of prolyl oligopeptidase (PO) elevates inositol phosphate (IP) signalling and reduces cell sensitivity to lithium (Li+). This review discusses recent evidence that shows PO acts via the multiple inositol polyphosphate phosphatase (MIPP) to regulate gene expression. As a consequence, PO inhibition causes both a transient, rapid increase in I(1,4,5)P(3) and a long-term elevation of IP signalling. This pathway is evolutionary conserved, being present in both the social amoeba Dictyostelium and human cell systems, and has potential implications for mental health.

m-Calpain activation is regulated by its membrane localization and by its binding to phosphatidylinositol 4,5-bisphosphate.

m-Calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP(2) points to a key regulatory role, although whether this PIP(2)-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP(2). Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP(2) is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP(2) specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.

A new role for PTEN in regulating transient receptor potential canonical channel 6-mediated Ca2+ entry, endothelial permeability, and angiogenesis.

Phosphatase and tensin homologue (PTEN) is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5-bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. We have uncovered a previously unrecognized role for PTEN in regulating Ca(2+) entry through transient receptor potential canonical channel 6 (TRPC6) that does not require PTEN phosphatase activity. We show that PTEN tail-domain residues 394-403 permit PTEN to associate with TRPC6. The inflammatory mediator thrombin promotes this association. Deletion of PTEN residues 394-403 prevents TRPC6 cell surface expression and Ca(2+) entry. However, PTEN mutant, C124S, which lacks phosphatase activity, did not alter TRPC6 activity. Thrombin failed to increase endothelial monolayer permeability in the endothelial cells, transducing the Δ394-403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing the Δ394-403 PTEN mutant. Our results demonstrate that PTEN, through residues 394-403, serves as a scaffold for TRPC6, enabling cell surface expression of the channel. Ca(2+) entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis. Thus, PTEN is indicated to play a role beyond suppressing PI3K signaling.