Michael J. Fry

Phosphatidylinositol 3-kinase : structure and expression of the 110 kd catalytic subunit

Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.

The GTPase dynamin binds to and is activated by a subset of SH3 domains

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3‐kinase (PI 3‐kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein‐serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N‐terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3‐kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter‐subunit serine phosphorylation in the regulation of the PI 3‐kinase in vivo.

Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C.

The Drosophila gene product Wingless (Wg) is a secreted glycoprotein and a member of the Wnt gene family. Genetic analysis of Drosophila epidermal development has defined a putative paracrine Wg signalling pathway involving the zeste‐white 3/shaggy (zw3/sgg) gene product. Although putative components of Wg‐ (and by inference Wnt‐) mediated signalling pathways have been identified by genetic analysis, the biochemical significance of most factors remains unproven. Here we show that in mouse 10T1/2 fibroblasts the activity of glycogen synthase kinase‐3 (GSK‐3), the murine homologue of Zw3/Sgg, is inactivated by Wg. This occurs through a signalling pathway that is distinct from insulin‐mediated regulation of GSK‐3 in that Wg signalling to GSK‐3 is insensitive to wortmannin. Additionally, Wg‐induced inactivation of GSK‐3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31–8220 and prolonged pre‐treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK‐3/Zw3 during Wnt/Wg signal transduction.

PI 3-kinase: structural and functional analysis of intersubunit interactions.

Phosphatidylinositol (PI) 3‐kinase has an 85 kDa subunit (p85 alpha) which mediates its association with activated protein tyrosine kinase receptors through SH2 domains, and an 110 kDa subunit (p110) which has intrinsic catalytic activity. Here p85 alpha and a related protein p85 beta are shown to form stable complexes with recombinant p110 in vivo and in vitro. Using a panel of glutathione S‐transferase (GST) fusion proteins of the inter‐SH2 region of p85, 104 amino acids were found to bind directly the p110 protein, while deletion mutants within this region further defined the binding site to a sequence of 35 amino acids. Transient expression of the mutant p85 alpha protein in mouse L cells showed it was unable to bind PI 3‐kinase activity in vivo. Mapping of the complementary site of interaction on the p110 protein defined 88 amino acids in the N‐terminal region of p110 which mediate the binding of this subunit to either the p85 alpha or the p85 beta proteins. The inter‐SH2 region of p85 is predicted to be an independently folded module of a coiled‐coil of two long anti‐parallel alpha‐helices. The predicted structure of p85 suggests a basis for the intersubunit interaction and the relevance of this interaction with respect to the regulation of the PI 3‐kinase complex is discussed.

The phosphoinositide (PI) 3-kinase family

![Figure][1] Phosphoinositide (PI) 3-kinase was first observed in 1984 as a minor inositol lipid kinase activity associated with immunoprecipitated oncogene products (e.g. Src, Abl and polyoma mT antigen) and present in activated growth factor receptor complexes (e.g. PDGF receptor). In 1988

Interaction of Axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription

Axin promotes the phosphorylation of β‐catenin by GSK‐3β, leading to β‐catenin degradation. Wnt signals interfere with β‐catenin turnover, resulting in enhanced transcription of target genes through the increased formation of β‐catenin complexes containing TCF transcription factors. Little is known about how GSK‐3β‐mediated β‐catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl‐2, a member of the Dishevelled family of proteins that function upstream of GSK‐3β. Expression of Dvl‐2 activated TCF‐dependent transcription. This was blocked by co‐expression of GSK‐3β or Axin. Expression of a 59 amino acid GSK‐3β‐binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full‐length Axin that prevented GSK‐3β binding also generated a transcriptional activator. When co‐expressed, Axin and Dvl‐2 co‐localized within expressing cells. When Dvl‐2 localization was altered using a C‐terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co‐immunoprecipitation data. Deletion analysis suggested that Dvl‐association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl‐2 may be important in the transmission of Wnt signals from Dvl‐2 to GSK‐3β.

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4‐phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5)P). We show that PtdIns(5)P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri‐induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short‐chain penetrating PtdIns(5)P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5)P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD‐expressing cells. Accordingly, IpgD and PtdIns(5)P production specifically activates a class IA PI 3‐kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3‐kinase/Akt activation via PtdIns(5)P production that plays an important role in host cell responses such as survival.

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.