Polyomavirus middle tumor antigen (MT) transform a large number of cell types by binding to and modulating the activities of cellular proteins. Previous genetic ansis defined in MT an independent motif, NPTY (Asn-Pro- Because expression of pp60v-ff induces tyrosine phosphorylation of SHC and subsequent activation of Ras, activated pp60c-src in complex with MT might do the same. A potential candidate for SHC had been reported in MT immunoprecipitates, a 51-kDa protein of unknown identity (34). Because phosphorylation at Tyr-250 was implicated in transformation, it was tempting to hypothesize that SHC might bind via its SH2 to this site. Our data confirm that SHC does bind to MT in an NPTY-dependent manner. MATERIALS AND METHODS 6344The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and plays a role in the transcriptional regulation of phospholipid synthesis genes. PAP is phosphorylated at multiple Ser and Thr residues and is dephosphorylated for in vivo function by the Nem1p-Spo7p protein phosphatase complex localized in the nuclear/endoplasmic reticulum membrane. In this work, we characterized seven previously identified phosphorylation sites of PAP that are within the Ser/Thr-Pro motif. When expressed on a low copy plasmid, wild type PAP could not complement the pah1⌬ mutant in the absence of the Nem1p-Spo7p complex. However, phosphorylation-deficient PAP (PAP-7A) containing alanine substitutions for the seven phosphorylation sites bypassed the requirement of the phosphatase complex and complemented the pah1⌬ nem1⌬ mutant phenotypes, such as temperature sensitivity, nuclear/endoplasmic reticulum membrane expansion, decreased triacylglycerol synthesis, and derepression of INO1 expression. Subcellular fractionation coupled with immunoblot analysis showed that PAP-7A was highly enriched in the membrane fraction. In fluorescence spectroscopy analysis, the PAP-7A showed tighter association with phospholipid vesicles than wild type PAP. Using site-directed mutagenesis of PAP, we identified Ser In the yeast Saccharomyces cerevisiae, the PAH1-encoded phosphatidate phosphatase (PAP) 2,3 catalyzes the dephosphorylation of PA, yielding DAG and P i (1, 2). This reaction is dependent on Mg 2ϩ ions and is based on a DXDX(T/V) catalytic motif within a haloacid dehalogenase-like domain in the enzyme (2-4). PAP is associated with the cytosolic and membrane fractions of the cell, and the association with the membrane is peripheral in nature (2). Chromatin immunoprecipitation analysis indicates that PAP is also localized in the nucleus (5). The DAG generated in the PAP reaction is used for the synthesis of TAG (2) and for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the CDPethanolamine and CDP-choline branches, respectively, of the Kennedy pathway (4, 6). The enzyme also plays a major role in controlling the cellular concentration of its substrate PA (2), the precursor of phospholipids that are synthesized via the CDP-DAG pathway (6 -8). In addition, the substrate PA plays a signaling role in the transcriptional regulation of phospholipid synthesis genes (9). In fact, mutants defective in PAH1-encoded PAP activity exhibit a Ͼ90% reduction in TAG content, a derepression of phospholipid synthesis genes, and an expansion of the nuclear/ER membrane (3, 5). Thus, the regulation of PAP activity governs the synthesis of TAG, the pathways by which phospholipids are synthesized, PA signaling, and the growth of the nuclear/ER membrane (6).The importance of PAP in lipid metabolism and cell physiology is further emphasized by the fact that the overexpression of Lpin1-encoded PAP (also known as lipin 1) in mice leads to obesity and insulin sensitivity, w...

Middle tumor antigen (MT) is the primary transforming protein of murine Polyomavirus. MT transforms by associating with and modulating the activities of cellular proteins involved in control of cell proliferation. MT binds to and is phosphorylated by cellular tyrosine kinases. The phosphorylated tyrosines become docking sites for SH2 (Src homology 2) domain-containing molecules. Tyrosine 322 of MT is known to be phosphorylated but has no known binding protein. We have found that phospholipase C-gamma 1 (PLC-gamma 1), a SH2 domain-containing protein, coimmunoprecipitates with MT. Tyrosine phosphorylation of PLC-gamma 1 is elevated in cells expressing MT, suggesting activation of this enzyme by MT. A Tyr-322-->Phe mutation in MT renders it defective in MT-PLC-gamma 1 interaction and in transformation. From the correlation between transformation and MT-PLC-gamma 1 interaction, we suggest that PLC-gamma 1 may play a role in transformation.

Background: Yeast Pah1 phosphatidate phosphatase required for triacylglycerol synthesis is subject to proteasome-mediated degradation. Results: Pah1 is degraded by the 20S proteasome in a ubiquitin-independent manner that is governed by its phosphorylation state. Conclusion: 20S proteasomal degradation of Pah1 is regulated by phosphorylation and dephosphorylation. Significance: Pah1 function in lipid metabolism is regulated by the 20S proteasome.

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time-and dose-dependent and was dependent on the concentrations of Pah1 (K m ‫؍‬ 0.23 M) and ATP (K m ‫؍‬ 5.5 M). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818.

This article is available online at http://www.jlr.org cause of mutations in the lpin1 gene ( 3, 6 ). The PA phosphatase reaction requires Mg 2+ ions and is based on a D X D X (T/V) catalytic motif within a haloacid dehalogenase-like domain in the enzyme ( 3, 7 ). The DAG produced by the enzyme is utilized for triacylglycerol (TAG) synthesis, and for the synthesis of the major phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC) ( 1,8 ). PA phosphatase also plays a major role in controlling the cellular content of its substrate PA ( 3 ), the precursor of phospholipids that are synthesized via the liponucleotide intermediate 8 ). In addition, PA plays a signaling role in the regulation of phospholipid synthesis gene expression ( 9 ).The importance of PA phosphatase in yeast is exemplifi ed by an assortment of pah1 ⌬ mutant phenotypes. For example, cells bearing the pah1 ⌬ mutation exhibit elevated levels of PA and reduced levels of DAG and TAG ( 3, 7 ). The elevated PA content causes the induction of phospholipid synthesis gene expression and the aberrant expansion of the nuclear/ER membrane ( 3, 7, 10 ), whereas the reduced capacity to synthesize DAG and TAG causes a defect in lipid droplet formation ( 11 ) and an acute sensitivity to fatty acid-induced toxicity ( 12 ), respectively. In addition, loss of PA phosphatase causes a respiratory defi ciency phenotype and sensitivity to growth at elevated temperature ( 3, 10 ).Mutations in mammalian lipins (counterpart of yeast PAH1 -encoded PA phosphatase) also cause defects in lipid metabolism and cell physiology. Mice lacking lipin 1 exhibit defects in adipose tissue development, lipodystrophy, insulin resistance, and peripheral neuropathy ( 4, 5, 13-16 ). In humans, lipin 1 mutations are associated with metabolic syndrome, type 2 diabetes, and recurrent acute myoglobinuria in children, whereas mutations in lipin 2 Abstract Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. The yeast Saccharomyces cerevisiae serves as a eukaryotic model to study the biochemistry and physiological roles of phosphatidate (PA) phosphatase ( 1 ), the enzyme that catalyzes the dephosphorylation of PA yielding diacylglycerol (DAG) and P i ( 2 ). The gene encoding the enzyme in yeast is PAH1 ( 3 ), and in fact, its discovery led to the revelation that the lipodystrophic defect in the fatty liver dystrophy ( fl d ) mouse ( 4, 5 ) was a PA phosphatase defi ciency be-

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Here we describe a reliable method for isolating genes that are differentially expressed in two cell populations. The method is a combination of subtractive hybridization and PCR. Among many improvements to previously described methods is the incorporation of a new technology into the procedure which sterilizes(inactivates) PCR amplicons, and thereby overcomes the limitation of similar procedures. To test this improved method, we conducted a search for estrogen-responsive genes. Estrogen-regulated genes dominated the subtracted libraries after four rounds of subtractive hybridizations. Four estrogen-regulated genes were identified from the initial screening.