The phosphatidylinositol 3-kinase (PI3K) signaling pathway is deregulated in many human diseases including cancer, diabetes, obesity, and autoimmunity. PI3K consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The p110-PI3K enzyme generates the key signaling lipid phosphatidylinositol 3,4,5-trisphosphate, which is dephosphorylated by the PI3-phosphatase PTEN. Here we show another function for the p85α regulatory protein: it binds directly to and enhances PTEN lipid phosphatase activity. We demonstrate that ectopically expressed FLAG-tagged p85 coimmunoprecipitates endogenous PTEN in an epidermal growth factor dependent manner. We also show epidermal growth factor dependent coimmunoprecipitation of endogenous p85 and PTEN proteins in HeLa cells. Thus p85 regulates both p110-PI3K and PTEN-phosphatase enzymes through direct interaction. This finding underscores the need for caution in analyzing PI3K activity because anti-p85 immunoprecipitations may contain both p85:p110-PI3K and p85:PTEN-phosphatase enzymes and thus measure net PI3K activity. We identify the N-terminal SH3-BH region of p85α, absent in the smaller p55α and p50α isoforms, as the region that mediates PTEN binding and regulation. Cellular expression of p85ΔSH3-BH results in substantially increased magnitude and duration of pAkt levels in response to growth factor stimulation. The ability of p85 to bind and directly regulate both p110-PI3K and PTEN-PI3-phosphatase allows us to explain the paradoxical insulin signaling phenotypes observed in mice with reduced PI3K or PTEN proteins. This discovery will impact ongoing studies using therapeutics targeting the PI3K/PTEN/Akt pathway.

This article is available online at http://www.jlr.org secretion under lipid-rich conditions. J. Lipid Res. 2010. 51: 150-161. Supplementary key words triacylglycerol • hypotriglyceridemia • hypertriglyceridemia • microsomal triglyceride transfer protein • hepatocyteApolipoprotein (apo) C-III is a small exchangeable apolipoprotein (79 amino acids) that is synthesized mainly in the liver and intestine from the APOA5-APOA4-APOC3-APOA1 gene locus and is secreted into the plasma as a component of VLDL and HDL ( 1 ). Elevated plasma apoC-III concentration is commonly observed in human subjects with insulin resistance and central obesity and is positively correlated with plasma triacylglycerol (TAG) concentrations in hypertriglyceridemia subjects ( 2, 3 ). Early studies have shown that mutations within the APOA4-APOC3-APOA1 gene locus were associated with patients of premature atherosclerosis ( 4, 5 ). However, the close proximity of apoA-IV, apoC-III, and apoA-I encoded within this gene cluster makes it diffi cult to ascertain the contribution of apoC-III defi ciency per se to the development of atherosclerosis. Recently, a genome-wide association study has discovered an apoC-III null allele (R19×) in Lancaster Amish population and shown that individuals heterozygous of the R19× allele have a favorable plasma lipid proAbstract Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion effi ciency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL 1 . Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL 1 secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identifi ed in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions. -Sundaram, M.,

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1a and 1b), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1a or -1b increased the synthesis and secretion of [ 3 H]glycerol-labeled lipids under either basal-or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL 1 (S f . 100) and VLDL 2 (S f 20-100). Expression of lipin-1a or -1b increased secretion efficiency and decreased intracellular degradation of [35 S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [ 3 H]glycerolipids and [ 35 S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1a not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1a mutant failed to promote

We studied the structural requirements of apolipoprotein (apo) B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB). Recombinant h-apoB48, like endogenous rat apoB48 (r-apoB48), was secreted as VLDL in addition to high density lipoproteins (HDL) by transfected cells, indicating that the N-terminal 48% of apoB contains sequences sufficient for VLDL assembly. Truncation of the C terminus of h-apo-B48 to -B42 or -B37 had little effect on the ability of apoB to assemble VLDL, whereas truncation to -B34 or -B29 markedly diminished or abolished VLDL formation. None of the truncations affected the integration of apoB into HDL. To determine whether the ability to assemble VLDL is governed by apoB length or by sequences beyond apoB29, we created chimeric proteins that contained human apoA-I and a segment derived from between the C-terminal 29 and 34%, 34 and 37%, or 37 and 42% of apoB100. The resulting chimeras, namely AI/B29 -34, AI/ B34 -37, and AI/B37-42, were secreted by the transfected cells as lipoproteins with buoyant density (d < 1.006 g/ml), electrophoretic mobility (pre-␤), and size characteristics of human plasma VLDL. The chimeras could assemble discrete VLDL particles devoid of endogenous r-apoB100, and could actively recruit triglycerides and phospholipids into the lipoproteins. However, these chimeras were secreted inefficiently. Pulse-chase analysis showed that less than 5% of the newly synthesized AI/B proteins were secreted, and more than 70% was degraded intracellularly. Degradation of the chimeras could be blocked by the cysteine protease inhibitor Nacetyl-leucyl-leucyl-norleucinal, but the treatment did not enhance their secretion. Protease protection analysis of microsomes isolated from transfected cells indicated that >65% of AI/B chimeras (compared with <25% of r-apoB100) were inaccessible to exogenous trypsin. These data suggest that the recruitment of large quantities of triglycerides during VLDL formation is not governed simply by apoB length, but is mediated by short hydrophobic sequences ranging from 152 to 237 amino acids (3-5%) of apoB. The existence of multiple such hydrophobic sequences within apoB48 may facilitate efficient assembly of hepatic VLDL particles.

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitopetagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.The low density lipoprotein receptor-related protein 1 (LRP1) 1 is a large type I membrane protein (4525 amino acids) and belongs to the low density lipoprotein receptor (LDLR) gene family (1, 2). Mammalian LRP1 is abundantly expressed in hepatocytes, neurons, smooth muscle cells, fibroblasts, and adipocytes. LRP1 is cleaved post-translationally by the endopeptidase furin in the distal Golgi, and the resulting extracellular ␣-chain (515 kDa) and transmembrane ␤-chain (85 kDa) form a non-covalent heterodimer (3). The ␣-chain is composed of three major structural modules, namely class A ligand-binding (complement-type) repeats, the epidermal growth factor (EGF) precursor-type repeats, and YWTD ␤-propellers. The ligand-binding repeats are dispersed throughout the LRP1 extracellular domain in four clusters denoted I to IV, of which clusters II and IV possess the ability to bind a variety of structurally unrelated ligands (4, 5). The cytoplasmic tail of the LRP1 ␤-chain contains motifs resembling those required for receptor-mediated endocytosis through clathrin-coated pits (e.g. NPXY and YXXL) that are found in other LDLR family members (6, 7). In addition, the cytoplasmic tail of th...

This article is available online at http://www.jlr.org secretion under lipid-rich conditions. J. Lipid Res. 2010. 51: 150-161. Supplementary key words triacylglycerol • hypotriglyceridemia • hypertriglyceridemia • microsomal triglyceride transfer protein • hepatocyteApolipoprotein (apo) C-III is a small exchangeable apolipoprotein (79 amino acids) that is synthesized mainly in the liver and intestine from the APOA5-APOA4-APOC3-APOA1 gene locus and is secreted into the plasma as a component of VLDL and HDL ( 1 ). Elevated plasma apoC-III concentration is commonly observed in human subjects with insulin resistance and central obesity and is positively correlated with plasma triacylglycerol (TAG) concentrations in hypertriglyceridemia subjects ( 2, 3 ). Early studies have shown that mutations within the APOA4-APOC3-APOA1 gene locus were associated with patients of premature atherosclerosis ( 4, 5 ). However, the close proximity of apoA-IV, apoC-III, and apoA-I encoded within this gene cluster makes it diffi cult to ascertain the contribution of apoC-III defi ciency per se to the development of atherosclerosis. Recently, a genome-wide association study has discovered an apoC-III null allele (R19×) in Lancaster Amish population and shown that individuals heterozygous of the R19× allele have a favorable plasma lipid proAbstract Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion effi ciency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL 1 . Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL 1 secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identifi ed in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions. -Sundaram, M.,

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