Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110 and are disrupted in oncogenic p85 mutants

Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 11/2009; 106(48):20258-63. DOI: 10.1073/pnas.0902369106
Source: PubMed

ABSTRACT We previously proposed a model of Class IA PI3K regulation in which p85 inhibition of p110alpha requires (i) an inhibitory contact between the p85 nSH2 domain and the p110alpha helical domain, and (ii) a contact between the p85 nSH2 and iSH2 domains that orients the nSH2 so as to inhibit p110alpha. We proposed that oncogenic truncations of p85 fail to inhibit p110 due to a loss of the iSH2-nSH2 contact. However, we now find that within the context of a minimal regulatory fragment of p85 (the nSH2-iSH2 fragment, termed p85ni), the nSH2 domain rotates much more freely (tau(c) approximately 12.7 ns) than it could if it were interacting rigidly with the iSH2 domain. These data are not compatible with our previous model. We therefore tested an alternative model in which oncogenic p85 truncations destabilize an interface between the p110alpha C2 domain (residue N345) and the p85 iSH2 domain (residues D560 and N564). p85ni-D560K/N564K shows reduced inhibition of p110alpha, similar to the truncated p85ni-572(STOP). Conversely, wild-type p85ni poorly inhibits p110alphaN345K. Strikingly, the p110alphaN345K mutant is inhibited to the same extent by the wild-type or truncated p85ni, suggesting that mutation of p110alpha-N345 is not additive with the p85ni-572(STOP) mutation. Similarly, the D560K/N564K mutation is not additive with the p85ni-572(STOP) mutant for downstream signaling or cellular transformation. Thus, our data suggests that mutations at the C2-iSH2 domain contact and truncations of the iSH2 domain, which are found in human tumors, both act by disrupting the C2-iSH2 domain interface.

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    • "Identification of the PI3K regulatory subunit p85 as an important molecule for starvationinduced PI3K feedback inhibition is consistent with a number of previous studies that have also defined roles for p85 subunits in negatively regulating PI3K activity. For example, mutations in p110 catalytic subunits are thought to induce cell transformation via abrogating the ability of p85 to negatively regulate PI3K activity (Wu et al., 2009), and monomeric p85 can form sequestration complexes in response to insulin stimulation to restrict phosphotyrosine from PI3K p85/p110 dimers (Luo et al., 2005). In addition, p85 has been shown to bind to and promote the activity of PTEN following stimulus-induced activation of PI3K as a means of feedback inhibition (Taniguchi et al., 2006). "
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    ABSTRACT: The IκB kinase (IKK) pathway is an essential mediator of inflammatory, oncogenic, and cell stress pathways. Recently IKK was shown to be essential for autophagy induction in mammalian cells independent of its ability to regulate NF-κB, but the mechanism by which this occurs is unclear. Here we demonstrate that the p85 regulatory subunit of PI3K is an IKK substrate, phosphorylated at S690 in vitro and in vivo in response to cellular starvation. Cells expressing p85 S690A or inhibited for IKK activity exhibit increased Akt activity following cell starvation, demonstrating that p85 phosphorylation is required for starvation-induced PI3K feedback inhibition. S690 is in a conserved region of the p85 cSH2 domain, and IKK-mediated phosphorylation of this site results in decreased affinity for tyrosine-phosphorylated proteins and decreased PI3K membrane localization. Finally, leucine deprivation is shown to be necessary and sufficient for starvation-induced, IKK-mediated p85 phosphorylation and PI3K feedback inhibition.
    Molecular cell 02/2012; 45(6):719-30. DOI:10.1016/j.molcel.2012.01.010 · 14.46 Impact Factor
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    • "Many of the iSH2 mutations are located in or closely adjacent to regions where we see an increase in exchange in the presence of lipid vesicles (D464H, D560Y, N564K/D, and L570P). Because we show that interaction with lipid vesicles causes a loosening between the iSH2 and p110δ, these mutations may mimic this membrane-mediated loosening and activate by disrupting the interaction between iSH2 and p110, as previously proposed (Wu et al., 2009). "
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    ABSTRACT: Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.
    Structure 08/2011; 19(8):1127-37. DOI:10.1016/j.str.2011.06.003 · 6.79 Impact Factor
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    • "However, subsequent studies on the nSH2-iSH2 fragment of p85α show that the nSH2 domain is in fact highly mobile with respect to the iSH2 domain. 15 N NMR relaxation methods were used to measure the rotational dynamics of the nSH2 domain within the nSH2-iSH2 construct, and defined an apparent rotational correlation time of 12.7 ± 0.7 ns for the nSH2 domain (Wu et al. 2009). These data are similar to experimental measurements for isolated SH2 domains (6.5–9.2 ns) and quite different from hydrodynamic calculations for several proposed nSH2-iSH2 geometries (45–52 ns) (Bernado et al. 2002; Farrow et al. 1994; Fushman et al. 1999; Zhang et al. 1998). "
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    ABSTRACT: Phosphoinositide 3-kinases (PI 3-kinases) are activated by growth factor and hormone receptors, and regulate cell growth, survival, motility, and responses to changes in nutritional conditions (Engelman et al. 2006). PI 3-kinases have been classified according to their subunit composition and their substrate specificity for phosphoinositides (Vanhaesebroeck et al. 2001). The class IA PI 3-kinase is a heterodimer consisting of one regulatory subunit (p85α, p85β, p55α, p50α, or p55γ) and one 110-kDa catalytic subunit (p110α, β or δ). The Class IB PI 3-kinase is also a dimer, composed of one regulatory subunit (p101 or p87) and one catalytic subunit (p110γ) (Wymann et al. 2003). Class I enzymes will utilize PI, PI[4]P, or PI[4,5]P2 as substrates in vitro, but are thought to primarily produce PI[3,4,5]P3 in cells.The crystal structure of the Class IB PI 3-kinase catalytic subunit p110γ was solved in 1999 (Walker et al. 1999), and crystal or NMR structures of the Class IA p110α catalytic subunit and all of the individual domains of the Class IA p85α regulatory subunit have been solved (Booker et al. 1992; Günther et al. 1996; Hoedemaeker et al. 1999; Huang et al. 2007; Koyama et al. 1993; Miled et al. 2007; Musacchio et al. 1996; Nolte et al. 1996; Siegal et al. 1998). However, a structure of an intact PI 3-kinase enzyme has remained elusive. In spite of this, studies over the past 10 years have lead to important insights into how the enzyme is regulated under physiological conditions. This chapter will specifically discuss the regulation of Class IA PI 3-kinase enzymatic activity, focusing on regulatory interactions between the p85 and p110 subunits and the modulation of these interactions by physiological activators and oncogenic mutations. The complex web of signaling downstream from Class IA PI 3-kinases will be discussed in other chapters in this volume.
    Current topics in microbiology and immunology 01/2010; 346(1):87-114. DOI:10.1007/82_2010_52 · 3.47 Impact Factor
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Ilker Sen