John Coadwell

The Gβγ Sensitivity of a PI3K Is Dependent upon a Tightly Associated Adaptor, p101

Two highly similar, PtdIns(4,5)P2-selective, G beta gamma-activated PI3Ks were purified from pig neutrophil cytosol. Both were heterodimers, were composed of a 101 kDa protein and either a 120 kDa or a 117 kDa catalytic subunit, and were activated greater than 100-fold by G beta gammas. Peptide sequence-based oligonucleotide probes were used to clone cDNAs for the p120 and p101 species. The cDNA of p120 is highly related to p110 gamma, while the cDNA of p101 is not substantially related to anything in current databases. The proteins were expressed in and purified from insect and mammalian cells. They bound tightly to one another, both in vivo and in vitro, and in so doing, p101 amplified the effect of G beta gammas on the PI3K activity of p120 from less than 2-fold to greater than 100-fold.

PtdIns(3)P Regulates the Neutrophil Oxidase Complex by Binding to the PX Domain of p40phox

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b558 (gp91phox and p22phox) and three soluble factors (GTP–Rac, p47phox and p67phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40phox has subsequently been discovered as a binding partner for p67phox (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40phox to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40phox. These results offer new insights into the roles for PI(3)Ks and p40phox in ROS formation and define a cellular ligand for the orphan PX domain.

Translocation of PDK-1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B

BACKGROUND Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. Here, we have investigated the effects of membrane localisation of PDK-1 on PKB activation. RESULTS Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation was sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurred upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhanced the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain which did not translocate to the membrane did not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activated PKB to the extent that it could not be activated further by PDGF. CONCLUSIONS In response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localises with PKB, significantly contributing to the scale of PKB activation.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

p84, a New Gβγ-Activated Regulatory Subunit of the Type IB Phosphoinositide 3-Kinase p110γ

Summary A variety of genetic and inhibitor studies have shown that phosphoinositide 3-kinase γ (PI3Kγ) plays an essential role in a number of physiological responses, including neutrophil chemotaxis, mast cell degranulation, and cardiac function [1–6]. PI3Kγ is currently thought to be composed of a p110γ catalytic subunit and a single regulatory subunit, p101. The binding of p110γ to p101 dramatically increases the activation of the complex by Gβγ subunits and, hence, is thought to be critical for the coupling of PI3Kγ to G protein coupled receptors [7–9]. Here, we characterize a new regulatory subunit for PI3Kγ. p84 is present in human, mouse, chicken, frog, and fugu genomes and is located beside the p101 locus. It is broadly expressed in cells of the murine immune system. Both recombinant and endogenous p84 bind p110γ specifically and with high affinity. Binding of p84 to p110γ substantially increases the ability of Gβγ to stimulate phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5) P 3 ) production both in vitro and in vivo. However, the p84/p110γ heterodimer is approximately 4-fold less sensitive to Gβγs than p101/p110γ. Endogenous murine p84 expression is substantially reduced in the absence of p110γ expression. We conclude that p110γ has two potential regulatory subunits in vivo, p84 and p101.

Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P3, the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gβγs) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gβγs, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kγ, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P3, activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kγ to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gβγs, p101 and the Ras–RBD interaction all have important roles in the regulation of PI(3)Kγ in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kγ effectors.

Identification of a Binding Site for Integrin αEβ7 in the N-terminal Domain of E-cadherin

The integrin αEβ7, which is predominantly expressed on mucosal T lymphocytes, has recently been shown to recognize the cell adhesion molecule, E-cadherin, on epithelial cells. We have carried out mutations on E-cadherin, involving domain deletions as well as substitutions of specific amino acids, in order to identify the sites recognized by the integrin. Binding of αEβ7 required the presence of the first two N-terminal domains of E-cadherin. Deletion of extracellular domains 3 and 4 or truncation of the cytoplasmic domain of E-cadherin had no consequence on integrin binding. Substitution of a glutamic acid in the BC loop of the Ig structure of the fist, N-terminal, domain of E-cadherin abrogated binding of αEβ7. This mutation did not appear to affect the conformation of the domain nor the pattern of expression of E-cadherin on the cell surface. Synthetic peptides encompassing the first domain of E-cadherin had very little inhibitory effect on the interaction with αEβ7. Our results highlight structural dissimilarities between recognition of E-cadherin by αEβ7 and recognition of other members of the IgSF by integrins and show that the heterophilic (integrin binding) and homophilic sites in the N-terminal domain of E-cadherin are distinct.

Isolation and characterization of two novel A20-like proteins

The transcription factor nuclear factor kappa B (NF-kappa B) plays a pivotal role in inflammatory processes through induction of adhesion molecules and chemokines. The zinc finger molecule A20 is an important negative regulator of NF-kappa B. The mechanism utilized by A20 is not fully understood, but A20 has been shown to bind to tumour-necrosis-factor-receptor-associated factor (TRAF) molecules, which are necessary for pro-inflammatory cytokine signalling. We report two novel genes, Cezanne (cellular zinc finger anti-NF-kappa B) and TRABID (TRAF-binding domain), with sequence similarity to A20. Co-immunoprecipitation studies indicated that TRAF6 was able to interact with both Cezanne and TRABID. In contrast, reporter gene experiments revealed a specific ability of Cezanne to down-regulate NF-kappa B. It is likely, therefore, that Cezanne participates in the regulation of inflammatory processes.

Studies on transcriptional regulation of the mucosal T‐cell integrin αEβ7 (CD103)

Integrin αEβ7 is expressed almost exclusively by mucosal T cells and mucosal dendritic antigen‐presenting cells (APCs) and is thought to be induced locally by transforming growth factor‐β (TGF‐β). In mice, mRNA for the αE subunit was found to be abundant in mucosal T cells but absent from other tissues. Exposure of a T‐cell line to TGF‐β strongly up‐regulated αE mRNA levels within 30 min, and nuclear run‐on experiments established that regulation occurred at the level of transcription. The organization of the human αE gene and a very closely linked novel gene, ELG, was determined. The αE promoter was tested in T cells and fibroblasts and functioned equally well in both cell types and did not confer TGF‐β responsiveness. Regions of the promoter providing enhancer activity and phorbol 12‐myristate 13‐acetate (PMA) responsiveness were identified by deletion studies. DNAse 1 hypersensitivity analysis of 36 kb of the αE gene revealed one hypersensitive site, found only in αE+ cells, located near the transcription start points. These results show that, unlike the situation with other integrins, lineage specificity and cytokine responsiveness of αE transcription are not conferred by the proximal promoter. Specificity may depend on distant control elements that have not yet been identified.

Expression of a testis-specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis

Actin‐capping proteins are ubiquitous components of mammalian cells. They are known to regulate the polymerization state of actin and hence indirectly control the activity of the cytoskeleton and cell shape. As part of our investigation into the molecular mechanisms that direct differentiation of a round spermatid into an elongating spermatozoa, we report on a testis‐specific 1.7‐kb transcript from rat testis with sequence similarities to the α subunit of actin‐capping proteins (ACPs) from somatic cells. The transcript contains a putative cAMP‐responsive motif (CREM) upstream of the initiation codon in the DNA sequence and is expressed postmeiotically, first appearing between 20 and 30 days of postnatal development. The primary amino acid sequence is 90% identical to that of a previously identified testis‐specific mouse protein, gsg3, both showing approximately 40% homology to the α subunit of somatic ACPs. An affinity‐purified polyclonal antibody to a synthetic peptide derived from the rat transcript identified a 32‐kDa protein on Western blots of testicular extracts. Indirect immunofluorescent localization of the protein on frozen sections of adult rat testis showed that it is intracellular and accumulates asymmetrically in the cytoplasm of round spermatids coincident with the position of the developing acrosome. This spatial expression parallels the distribution of F‐actin during sperm differentiation, supporting the hypothesis that testis‐specific ACPs have an important role in determining the final shape of mature sperm heads. A disturbance in the expression of these ACPs may underlie many of the abnormalities in sperm morphology observed in infertile semen. Mol. Reprod. Dev. 49:81–91, 1998.