P E Rose

Dana-Farber Cancer Institute, Boston, Massachusetts, United States

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Publications (6)29.64 Total impact

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    ABSTRACT: Cytokines regulate the development and differentiated functions of hematopoietic cells by activating multiple signaling pathways, including the Jak-Stat pathway, the PI3-kinase pathway, and the Ras/Raf pathway. While the Jak-Stat interaction has been extensively studied, the relationship between this pathway and other cytokine-induced signaling pathways is not fully understood. In Drosophila melanogaster, mutations that result in hyperactivity of the Jak kinase Hopscotch (Hop) cause an activation of the larval blood cell encapsulation response, including blood cell aggregation and differentiation of plasmatocytes into apparent lamellocytes. Here, we demonstrate that Hop requires the activity of the Raf pathway to promote the activation response of larval plasmatocytes, and provide evidence to suggest that the Hop and D-Raf proteins physically interact. We also show that basal level activity of the Raf pathway is required for the accumulation of circulating blood cells.
    Molecular and General Genetics 04/2002; 267(1):57-63. · 2.88 Impact Factor
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    ABSTRACT: The plethora of extracellular stimuli modulating the status of a cell results in the engagement of relatively few pathways responsible for transducing signals to the interior of the cell. One such pathway is the activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositide 3'-phosphate. Among the enzymes activated by 3'-phosphorylated inositol lipids is Akt/protein kinase B (PKB). Here we describe a protocol for the expression and one-step purification of human recombinant GST-PKB in Sf9 cells. This scheme allows generating large amounts of homogeneously purified GST-PKB with high specific activity to be employed in high-throughput screening or structural studies.
    Protein Expression and Purification 11/1999; 17(1):83-8. · 1.43 Impact Factor
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    ABSTRACT: Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.
    Journal of Virology 02/1998; 72(1):558-63. · 5.08 Impact Factor
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    ABSTRACT: The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine signal transduction pathways. In Drosophila melanogaster, the dominant hop(Tum-l) mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. Previous studies have suggested that the Hop(Tum-l) protein might be a hyperactive kinase. Here, we report on the new dominant mutation hop(T42), which causes abnormalities that are similar to but more extreme than those caused by hop(Tum-l). We determined that Hop(T42) contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. We determined that Hop(Tum-1) and Hop(T42) both hyperphosphorylated and hyperactivated D-Stat when overexpressed in Drosophila cells. Moreover, we found that the hop(T42) phenotype was partially rescued by a reduction of wild-type D-stat activity. Finally, generation of the corresponding E695K mutation in murine Jak2 resulted in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. Finally, we propose a model for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis.
    Molecular and Cellular Biology 04/1997; 17(3):1562-71. · 5.37 Impact Factor
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    P E Rose, B S Schaffhausen
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    ABSTRACT: Polyomavirus large tumor antigen (LT) contains a potential C2H2 zinc binding element between residues 452 and 472. LT also contains a third histidine in this region, conserved among the polyomavirus LTs. Synthetic peptides of this region bound a single atom of zinc, as determined by spectroscopic analysis. Blotting experiments also showed that fusion proteins containing the element, as well as full-length LT, bound 65Zn. Polyomavirus middle T and small T antigens also bound zinc in the blotting assay. Site-directed mutagenesis showed the importance of this element in LT. Point mutations in four of the conserved residues (C-452, C-455, H-465, and H-469) blocked the ability of LT to function in viral DNA replication, while mutation of H-472-->L decreased replication to 1/30th that of the wild type. Point mutations in intervening residues tested had little effect on replication. Mutants resulting from mutations in the conserved cysteine or histidine residues retained the ability to bind origin DNA. However, they did show a defect in self-association. Because double-hexamer formation is involved in DNA replication, this deficiency is sufficient to explain the defect in replication. Mutants created by point mutations of the coordinating residues were also deficient in replication-associated phosphorylations.
    Journal of Virology 06/1995; 69(5):2842-9. · 5.08 Impact Factor
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    ABSTRACT: Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation.
    Proceedings of the National Academy of Sciences 01/1995; 91(25):12125-9. · 9.81 Impact Factor