PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed ‘tandem-PH-domain-containing protein-1’ (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed ‘MUPP1’, a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2.

OBJECTIVE-Wnt signaling inhibits adipogenesis, but its regulation, physiological relevance, and molecular effectors are poorly understood. Here, we identify the Wnt modulator Dapper1/Frodo1 (Dact1) as a new preadipocyte gene involved in the regulation of murine and human adipogenesis. RESEARCH DESIGN AND METHODS-Changes in Dact1expression were investigated in three in vitro models of adipogenesis. In vitro gain-and loss-of-function studies were used to investigate the mechanism of Dact1 action during adipogenesis. The in vivo regulation of Dact1 and Wnt/␤-catenin signaling were investigated in murine models of altered nutritional status, of pharmacological stimulation of in vivo adipogenesis, and during the development of dietary and genetic obesity.

PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.

It has been postulated that PtdIns(3,4) P (2), one of the immediate breakdown products of PtdIns(3,4,5) P (3), functions as a signalling molecule in insulin- and growth-factor-stimulated pathways. To date, the t andem- P H-domain-containing p rotein- 1 (TAPP1) and related TAPP2 are still the only known PH-domain-containing proteins that interact strongly and specifically with PtdIns(3,4) P (2). In this study we demonstrate that endogenously expressed TAPP1, is constitutively associated with the protein-tyrosine-phosphatase-like protein-1 (PTPL1 also known as FAP-1). We show that PTPL1 binds to TAPP1 and TAPP2, principally though its first PDZ domain [where PDZ is postsynaptic density protein ( P SD-95)/ Drosophila disc large tumour suppressor ( d lg)/tight junction protein ( Z O1)] and show that this renders PTPL1 capable of associating with PtdIns(3,4) P (2) in vitro. Our data suggest that the binding of TAPP1 to PTPL1 does not influence PTPL1 phosphatase activity, but instead functions to maintain PTPL1 in the cytoplasm. Following stimulation of cells with hydrogen peroxide to induce PtdIns(3,4) P (2) production, PTPL1, complexed to TAPP1, translocates to the plasma membrane. This study provides the first evidence that TAPP1 and PtdIns(3,4) P (2) could function to regulate the membrane localization of PTPL1. We speculate that if PTPL1 was recruited to the plasma membrane by increasing levels of PtdIns(3,4) P (2), it could trigger a negative feedback loop in which phosphoinositide-3-kinase-dependent or other signalling pathways could be switched off by the phosphatase-catalysed dephosphorylation of receptor tyrosine kinases or tyrosine phosphorylated adaptor proteins such as IRS1 or IRS2. Consistent with this notion we observed RNA-interference-mediated knock-down of TAPP1 in HEK-293 cells, enhanced activation and phosphorylation of PKB following IGF1 stimulation.

We previously reported a family in which a heterozygous missense mutation in Akt2 led to a dominantly inherited syndrome of insulin-resistant diabetes and partial lipodystrophy. To determine whether genetic variation in AKT2 plays a broader role in human metabolic disease, we sequenced the entire coding region and splice junctions of AKT2 in 94 unrelated patients with severe insulin resistance, 35 of whom had partial lipodystrophy. Two rare missense mutations (R208K and R467W) were identified in single individuals. However, insulin-stimulated kinase activities of these variants were indistinguishable from wild type. In two large case-control studies (total number of participants 2,200), 0 of 11 common single nucleotide polymorphism (SNPs) in AKT2 showed significant association with type 2 diabetes. In a quantitative trait study of 1,721 extensively phenotyped individuals from the U.K., no association was found with any relevant intermediate metabolic trait. In summary, although heterozygous loss-offunction mutations in AKT2 can cause a syndrome of severe insulin resistance and lipodystrophy in humans, such mutations are uncommon causes of these syndromes. Furthermore, genetic variation in and around the AKT2 locus is unlikely to contribute significantly to the risk of type 2 diabetes or related intermediate metabolic traits in U.K. populations.

PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed 'tandem-PH-domain-containing protein-1' (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed 'MUPP1', a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2.

No abstract

The protein kinase C (PKC) superfamily have been implicated in the control of multiple cellular functions. This has been associated with their acute control (classical and novel PKC isotypes) through the production of the second messenger diacylglycerol (DAG). More recently however it has become clear that these proteins integrate controls from multiple sources. This regulation seems to involve not just phospholipase C (and DAG), but phosphatidylinos-itol3-kinase and mTOR pathways. These controls act together to produce fully active, phosphorylated forms of these PKC isotypes. The strategies employed by cells to control these events are diverse and can involve both agonist-dependent responses as well as cell-cell and/or cell-matrix dependent events. The relationship between cellmatrix associations and PKC alpha is of particularly interest. This isotype has profound effects upon integrin behaviour and integrindependent cellular migration. The integrin control of PKC family function and the PKC control of integrin function will be discussed.The interaction of insulin or growth factors with its receptor on the outside surface of a cell leads to the generation of a lipid 'second messenger' phospatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) at the inner surface of the cell membrane. I will describe our studies aimed at understanding the molecular mechanism by which PtdIns(3,4,5)P3 can activate signal transduction pathways that ultimately control the key physiological responses regulated by insulin and growth factors. I will present our initial work characterising the properties of the novel proteins termed DAPP1, TAPPl and T A W 2 that we and others have recently identified which interact with PtdIns(3,4,5)P3 and/or its immediate breakdown product PtdIns(3,4)P2. Another key intracel-M a r target of PtdIns(3,4,5)P3 , are a group of protein kinases, termed the A G C kinases. These include isoforms of protein kinase B (PKB/Akt), p70 ribosomal S6K (S6K) and protein kinase C (PKC) which are activated following PtdIns(3,4,5)P3 production in cells.. I will discuss some recent studies that we have performed on characterising the molecular mechanism by which these kinases are activated following their phosohorylation by the 3-phosphoinositide dependent protein kinase-l (PDKl). I will also present some preliminary findings on our studies on mice that are deficient in PDKl expression.D14 Tec family kinases in T cell receptor signaling NHGRI, N I H , 49/4A38,