Assembly and structure of the p85a homodimer
MetadataShow full item record
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3'hydroxyl in the inositol ring of phosphoinositides to generate the biologically active moiety PI(3,4,5)P3. PI3Ks are activated by growth factor and G-protein coupled receptors, and propagate intracellular signals for growth, survival, proliferation, and metabolism. Class IA PI3Ks are heterodimers consisting of one 110-kDa catalytic subunit (p110alpha, p110beta, or p110delta) and one regulatory subunit of 85, 55 or 50 kDa (p85alpha, p853, p55alpha, p55gamma, or p50alpha). p85alpha binds, stabilizes, and inhibits the enzymatic activity of p110alpha, beta, and delta, and tyrosine-phosphorylated peptides bind the N- and C-terminal SH2 domains of p85alpha, relieving its inhibitory effect on p110.;p85alpha is a modular protein consisting of 5 domains, the individual structures for which have all been solved. The N-terminus contains a Src homology 3 (SH3) domain flanked by two proline-rich motifs (PR1 and PR2). Next is a BCR homology domain followed by a coiled-coil inter SH2 (iSH2) domain that is flanked by two smaller SH2 domains (nSH2 and cSH2). The N-terminal region of p85alpha (SH3-BCR) does not interact with p110 and is relatively devoid of structural information, while the C-terminal half of the protein (nSH2-iSH2-cSH2) interacts with p110 and regions of this polypeptide have been co-crystallized with p110alpha and p110beta.;p85alpha homodimerizes via intermolecular contacts between its SH3-PR1 and BCR domains in addition to heterodimerizing with p110. Because p85alpha plays a central role in regulating the PI3K signaling pathway, characterization of its structure and assembly dynamics is crucial to understanding its function in normal physiology and disease. This study combines in vivo and in vitro solution characterization of p85alpha dimerization with modeling of its global architecture. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85alpha that expresses robustly in bacteria. This protein is readily purified in high quantities and is functionally equivalent to native p85alpha, as determined by assaying the function of each cysteine-free domain, as well as the ability of full-length cysteine-free p85alpha to bind and inhibit p110alpha activity.;Analytical ultracentrifugation (AUC) studies showed that p85alpha undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second interaction mediated by cSH2 at the C-terminal end of the polypeptide with unique electrostatic character. We have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle X-ray scattering (SAXS) and chemical cross-linking structural analysis of the discrete dimer. These experimental measures are used as constraints for structural modeling of the p85alpha dimer. Additionally, we have demonstrated in vivo concentration-dependent dimerization of p85alpha using fluorescence fluctuation spectroscopy (FFS). Our study provides new insight into the structure and assembly of the p85alpha dimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to efficiently exchange among multiple binding partners.;Additionally, we have developed a strategy to explore the spatial organization of p85alpha domains in the context of the p85alpha/p110alpha heterodimer using site-specific fluorescent labeling of p85alpha and analysis by single-molecule FRET (smFRET). This study will generate a spatial map of p85alpha domains within the heterodimer and allow measurement of changes in domain orientation upon binding of regulatory proteins that activate p85alpha/p110alpha. In addition to a fully functional cysteine-free p85alpha, we have constructed a mutant of p110alpha in which 17 surface cysteine residues have been mutated to prevent their labeling with maleimide-conjugated fluorescent dyes. Pairs of cysteine residues have been inserted at specific sites within p85alpha domains. By co-expression of p85alpha variants with single cysteine residues with cysteine-free p110alpha, we aim to produce dye-labeled complexes that can be used to map the relative distances between the domains of p85alpha to generate a set of distance constraints for structural modeling of p85alpha under basal and stimulated conditions. These experiments will potentially reveal the conformational changes that occur in p85alpha upon PI3K activation.;A recently described p85alpha binding partner is phosphatase and tensin homolog deleted on chromosome ten (PTEN), a lipid phosphatase that negatively regulates PI3 kinase signaling by dephosphorylating the products of PI3K. The N-terminus of p85alpha binds PTEN, protecting it from degradation and decreasing net PI3K signaling in the cell. A p85alpha fragment lacking the domains involved in dimerization (SH3 and BCR) was not able to bind PTEN, indicating that p85alpha dimerization may affect this regulatory interaction. To address this question, we have begun to bacterially express and purify recombinant PTEN for use in in vitro structure and assembly studies examining the p85alpha-PTEN association. We describe experiments that will explore the effect of p85alpha self-association on its ability to scaffold with PTEN and other regulatory partners.;Overall, our studies provide innovative ways of examining facets of p85alpha structure that have thus far proved elusive. Given its role as a scaffold in several major signaling pathways, it is important to understand how p85 shuttles between binding partners and participates in various complexes in different cellular contexts. The relative dearth of information regarding the region of p85alpha involved in its homodimerization in particular represents an opportunity for novel insight into our understanding of its role in multiple signaling pathways and protein interactions.
- Theses and Dissertations