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A structural and functional analysis of the phosphatidylinositol 3-kinase

Dhand, Ritu Bala; (1994) A structural and functional analysis of the phosphatidylinositol 3-kinase. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Phosphatidylinositol 3-kinase has emerged as an important signal transducing molecule which is activated by diverse receptor and non-receptor protein-tyrosine kinases (PTK) and in some cells may be an important mediator in the events leading to mitogenesis. The affinity purification of PI 3-kinase from bovine brain has shown that this enzyme is a tightly associated heterodimer of 85 kDa and 110 kDa subunits. Structural analysis at the protein and cDNA levels revealed two forms of the 85 kDa subunit, p85 [alpha] which corresponds to the material purified from the bovine brain, and another highly related protein termed p85 [beta]. Both p85 proteins contain an N-terminal SH3 domain, followed by a BCR domain and two SH2 domains separated by a region predicted to adopt a helical conformation. The p85 proteins have been expressed in insect cells using the baculovirus expression system and they are shown to form tight complexes with many PTKs when coexpressed in the same cell. The cloning of the catalytic bovine pi 10 subunit of PI 3-kinase has allowed expression of the p110 subunit in insect cells. This protein was subsequently shown to possess an intrinsic PI 3-kinase activity when expressed alone and to form a stable active complex with p85aand p85?both in vitro and in vivo. The sites of interaction between the two subunits have been mapped to the N-terminal region of pi 10 to a sequence of 35 amino acids in the inter-SH2 domain of p85. The p85[alpha]/p110 and the p85[beta]/p110 complexes bind to activated PTKs in a manner that is more selective than the binding of p85[alpha] or p85 [beta] alone to PTKs. Additional biochemical and mutagenesis studies of the catalytic p110 subunit have demonstrated that this is a novel dual specificity enzyme with an intrinsic protein-serine kinase activity which in turn regulates its PI 3-kinase activity. These studies using the recombinant enzyme have been complemented by the analysis of endogenous PI 3-kinase activation in PDGF-stimulated NIH 3T3 cells.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: A structural and functional analysis of the phosphatidylinositol 3-kinase
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10098263
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