Article

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α 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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    • "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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    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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Ilker Sen