P110?

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Lewis T. Williams - One of the best experts on this subject based on the ideXlab platform.

  • membrane localization of phosphatidylinositol 3 kinase is sufficient to activate multiple signal transducing kinase pathways
    Molecular and Cellular Biology, 1996
    Co-Authors: Anke Klippel, Christoph Reinhard, W M Kavanaugh, Gerald Apell, M A Escobedo, Lewis T. Williams
    Abstract:

    Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic P110? subunit to activated growth factor receptors and is required for enzymatic activity of P110?. In this report, we describe the effects of expressing novel forms of P110? that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized P110? is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of P110? (P110?*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of P110? or a form of P110? with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.

  • Activated Phosphatidylinositol 3-Kinase Is Sufficient to Mediate Actin Rearrangement and GLUT4 Translocation in 3T3-L1 Adipocytes
    Journal of Biological Chemistry, 1996
    Co-Authors: Stuart S. Martin, Tetsuro Haruta, Aaron J Morris, Anke Klippel, Lewis T. Williams, Jerrold M. Olefsky
    Abstract:

    Abstract Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane. Both processes depend on the activity of phosphatidylinositol 3-kinase. Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, P110?*, in 3T3-L1 adipocytes. Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of P110?* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles. These effects are inhibited by wortmannin in the P110?*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, P110?*Δkin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.

  • ras dependent induction of cellular responses by constitutively active phosphatidylinositol 3 kinase
    Science, 1995
    Co-Authors: Anke Klippel, Anthony J Muslin, Wendy J Fantl, Lewis T. Williams
    Abstract:

    Phosphatidylinositol (Pl)-3 kinase is one of many enzymes stimulated by growth factors. A constitutively activated mutant, P110?, that functions independently of growth factor stimulation was constructed to determine the specific responses regulated by Pl-3 kinase. The P110? protein exhibited high specific activity as a Pl-3 kinase and as a protein kinase. Expression of P110? in NIH 3T3 cells induced transcription from the fos promoter. Co-expression of dominant negative Ras blocked this response. When expressed in Xenopus laevis oocytes, P110? increased the amount of guanosine 5'-triphosphate-bound Ras, caused activation of the Ras effector Raf-1, and induced Ras-dependent oocyte maturation. These findings show that Pl-3 kinase can stimulate diverse Ras-dependent cellular processes, including oocyte maturation and fos transcription.

  • the interaction of small domains between the subunits of phosphatidylinositol 3 kinase determines enzyme activity
    Molecular and Cellular Biology, 1994
    Co-Authors: Anke Klippel, Jaime Escobedo, M Hirano, Lewis T. Williams
    Abstract:

    Abstract Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and P110?, function as localizing and catalytic subunits, respectively. Using recombinant p85 and P110? molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in P110? which directs the binding to p85. We demonstrate that the interactive domains in p85 and P110? are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and P110? mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length P110? molecule is catalytically active, whereas P110? alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of P110? were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the P110? catalytic subunit, is an important component of the catalytic complex.

Anke Klippel - One of the best experts on this subject based on the ideXlab platform.

  • membrane localization of phosphatidylinositol 3 kinase is sufficient to activate multiple signal transducing kinase pathways
    Molecular and Cellular Biology, 1996
    Co-Authors: Anke Klippel, Christoph Reinhard, W M Kavanaugh, Gerald Apell, M A Escobedo, Lewis T. Williams
    Abstract:

    Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic P110? subunit to activated growth factor receptors and is required for enzymatic activity of P110?. In this report, we describe the effects of expressing novel forms of P110? that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized P110? is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of P110? (P110?*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of P110? or a form of P110? with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.

  • Activated Phosphatidylinositol 3-Kinase Is Sufficient to Mediate Actin Rearrangement and GLUT4 Translocation in 3T3-L1 Adipocytes
    Journal of Biological Chemistry, 1996
    Co-Authors: Stuart S. Martin, Tetsuro Haruta, Aaron J Morris, Anke Klippel, Lewis T. Williams, Jerrold M. Olefsky
    Abstract:

    Abstract Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane. Both processes depend on the activity of phosphatidylinositol 3-kinase. Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, P110?*, in 3T3-L1 adipocytes. Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of P110?* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles. These effects are inhibited by wortmannin in the P110?*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, P110?*Δkin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.

  • ras dependent induction of cellular responses by constitutively active phosphatidylinositol 3 kinase
    Science, 1995
    Co-Authors: Anke Klippel, Anthony J Muslin, Wendy J Fantl, Lewis T. Williams
    Abstract:

    Phosphatidylinositol (Pl)-3 kinase is one of many enzymes stimulated by growth factors. A constitutively activated mutant, P110?, that functions independently of growth factor stimulation was constructed to determine the specific responses regulated by Pl-3 kinase. The P110? protein exhibited high specific activity as a Pl-3 kinase and as a protein kinase. Expression of P110? in NIH 3T3 cells induced transcription from the fos promoter. Co-expression of dominant negative Ras blocked this response. When expressed in Xenopus laevis oocytes, P110? increased the amount of guanosine 5'-triphosphate-bound Ras, caused activation of the Ras effector Raf-1, and induced Ras-dependent oocyte maturation. These findings show that Pl-3 kinase can stimulate diverse Ras-dependent cellular processes, including oocyte maturation and fos transcription.

  • the interaction of small domains between the subunits of phosphatidylinositol 3 kinase determines enzyme activity
    Molecular and Cellular Biology, 1994
    Co-Authors: Anke Klippel, Jaime Escobedo, M Hirano, Lewis T. Williams
    Abstract:

    Abstract Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and P110?, function as localizing and catalytic subunits, respectively. Using recombinant p85 and P110? molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in P110? which directs the binding to p85. We demonstrate that the interactive domains in p85 and P110? are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and P110? mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length P110? molecule is catalytically active, whereas P110? alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of P110? were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the P110? catalytic subunit, is an important component of the catalytic complex.

Albrecht Bindereif - One of the best experts on this subject based on the ideXlab platform.

  • human u4 u6 snrnp recycling factor P110? mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling
    Molecular and Cellular Biology, 2004
    Co-Authors: Jan Medenbach, Silke Schreiner, Sunbin Liu, Reinhard Lührmann, Albrecht Bindereif
    Abstract:

    After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein P110? (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-P110? is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that P110? interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with P110?. Together, these data suggest a model whereby P110? brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

  • P110? a novel human u6 snrnp protein and u4 u6 snrnp recycling factor
    The EMBO Journal, 2002
    Co-Authors: Mathias Bell, Andrey Damianov, Silke Schreiner, Ram Reddy, Albrecht Bindereif
    Abstract:

    During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4–U6 and U6–U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as P110?nrb or SART3. P110? contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of P110?, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. P110? homologs sharing the same domain structure also exist in several other eukaryotes. P110? is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri-snRNPs nor with spliceosomes. Recom binant P110? binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that P110? functions in the reassembly of the U4/U6 snRNP. In summary, P110? represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.

Michael D Waterfield - One of the best experts on this subject based on the ideXlab platform.

  • impaired b and t cell antigen receptor signaling in P110?delta pi 3 kinase mutant mice
    Science, 2002
    Co-Authors: Klaus Okkenhaug, Antonio Bilancio, Geraldine Farjot, Helen Priddle, Sara Sancho, Emma Peskett, Wayne Pearce, Stephen Meek, Ashreena Salpekar, Michael D Waterfield
    Abstract:

    Class IA phosphoinositide 3-kinases (PI3Ks) are a family of p85/P110? heterodimeric lipid kinases that generate second messenger signals downstream of tyrosine kinases, thereby controlling cell metabolism, growth, proliferation, differentiation, motility, and survival. Mammals express three class IA catalytic subunits: P110?alpha, P110?beta, and P110?delta. It is unclear to what extent these P110? isoforms have overlapping or distinct biological roles. Mice expressing a catalytically inactive form of P110?delta (P110?delta(D910A)) were generated by gene targeting. Antigen receptor signaling in B and T cells was impaired and immune responses in vivo were attenuated in P110?delta mutant mice. They also developed inflammatory bowel disease. These results reveal a selective role for P110?delta in immunity.

  • the drosophila phosphoinositide 3 kinase dP110? promotes cell growth
    The EMBO Journal, 1996
    Co-Authors: Sally J Leevers, David Weinkove, Lindsay K Macdougall, E Hafen, Michael D Waterfield
    Abstract:

    Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium. They are activated by a multitude of extracellular signals and implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape. Here we describe the molecular and functional analysis of Drosophila P110? (DP110?). A full-length DP110? cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks P110?alpha and P110?beta. Overexpression of DP110? in wing or eye imaginal discs resulted in flies with enlarged wings or eyes respectively. In contrast, overexpression of DP110? containing a mutation predicted to result in the loss of catalytic activity resulted in smaller wings and eyes. The alterations in wing size result from changes in both cell size and cell number, whereas in the eye only differences in cell size were detected. These data imply a role for DP110? in growth control during Drosophila development and have implications for the function of class I PI3Ks in other organisms.

  • structure and function of phosphatidylinositol 3 kinase a potential second messenger system involved in growth control
    Philosophical Transactions of the Royal Society B, 1993
    Co-Authors: Michael J Fry, Michael D Waterfield
    Abstract:

    Ligand stimulation of growth factor receptors with intrinsic protein-tyrosine kinase activity initiates the assembly of multienzyme signalling complexes. This is mediated by binding of proteins with src homology 2 (SH2) domains to receptor autophosphorylation sites. Among the proteins involved in complex formation is phosphatidylinositol (PI) 3-kinase, a heterodimeric enzyme composed of 85 kDa and 110 kDa subunits, which binds to receptor (and non-receptor) phosphotyrosine residues through the two SH2 domains in the p85 subunit. p85 acts as an adaptor protein and possibly a regulator of the P110? catalytic subunit that phosphorylates phosphoinositides at the D-3 position of the inositol ring. p85 subunit is composed of several distinct functional domains: one SH3 and two SH2 domains, a P110? binding site and a region with homology to BCR. Expression of these domains in E. coli as GST-fusion proteins has allowed definition by nuclear magnetic resonance (NMR) of three-dimensional structures for the SH2 and SH3 domains. The relationship of structure to function for these domains is discussed. The P110? catalytic domain has a region of homology with vps34p of Saccharomyces cerevisiae, a protein involved in protein sorting to the yeast vacuole. Possible clues to the function of PI 3-kinase derived from this and other observations are presented.

Jerrold M. Olefsky - One of the best experts on this subject based on the ideXlab platform.

  • Activated Phosphatidylinositol 3-Kinase Is Sufficient to Mediate Actin Rearrangement and GLUT4 Translocation in 3T3-L1 Adipocytes
    Journal of Biological Chemistry, 1996
    Co-Authors: Stuart S. Martin, Tetsuro Haruta, Aaron J Morris, Anke Klippel, Lewis T. Williams, Jerrold M. Olefsky
    Abstract:

    Abstract Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane. Both processes depend on the activity of phosphatidylinositol 3-kinase. Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, P110?*, in 3T3-L1 adipocytes. Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of P110?* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles. These effects are inhibited by wortmannin in the P110?*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, P110?*Δkin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.