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: 9.67). 11/2009; 106(48):20258-63. DOI: 10.1073/pnas.0902369106
Source: PubMed


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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    • "We have shown previously that the helical domain mutations E542K and E545K are by far the most common mutations found in PIK3CA in bladder cancer [4] and are more potent than H1047R in inducing signaling downstream of AKT and proliferation at confluence and under conditions of nutrient depletion when expressed in normal urothelial cells [29]. As the R358X truncation mutant removes both iSH2 and cSH2 regions of the protein, interaction with the C2 domain of p110α is lost and effects may mimic those described for mutations and truncations affecting these domains [33]. From sequencing data, and knowledge that there was minimal contaminating normal tissue in the sample, this mutation appeared to be heterozygous. "
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    ABSTRACT: Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.
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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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    Preview · Article · Feb 2012 · Molecular cell
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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.
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