Lipopolysaccharide activates plasma-membrane signaling and endosomal signaling by Toll-like receptor 4 (TLR4) through the TIRAP-MyD88 and TRAM-TRIF adaptor complexes, respectively, but it is unclear how the signaling switch between these cell compartments is coordinated. In dendritic cells, we found that the p110δ isoform of phosphatidylinositol-3-OH kinase (PI(3)K) induced internalization of TLR4 and dissociation of TIRAP from the plasma membrane, followed by calpain-mediated degradation of TIRAP. Accordingly, inactivation of p110δ prolonged TIRAP-mediated signaling from the plasma membrane, which augmented proinflammatory cytokine production while decreasing TRAM-dependent endosomal signaling that generated antiinflammatory cytokines (interleukin 10 and interferon-β). In line with that altered signaling output, p110δ-deficient mice showed enhanced endotoxin-induced death. Thus, by controlling the 'topology' of TLR4 signaling complexes, p110δ balances overall homeostasis in the TLR4 pathway.

Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110␣, p110␤, or p110␦) bound to any of five distinct regulatory subunits (p85␣, p85␤, p55␥, p55␣, and p50␣, collectively referred to as ''p85s''). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85␣ has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000 -15,000 p85/p110 complexes per cell, with p110␤ and p110␦ being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms.quantitative mass spectrometry ͉ signaling ͉ protein stability ͉ gene knockout ͉ lipid kinase P hosphoinositide 3-kinases (PI3Ks) generate lipid second messengers that serve as membrane docking sites for a variety of downstream effector proteins such as protein kinases, regulators of small GTPases, and scaffolding proteins (1, 2). The class IA PI3Ks are heterodimers consisting of a p110 catalytic subunit and a smaller regulatory subunit with Src-homology 2 (SH2) domains. Mammals have three catalytic subunits (p110␣, p110␤, p110␦) and five regulatory subunits (p85␣, p85␤, p55␥, p55␣, p50␣) (1, 2). Under experimental conditions, each p110 isoform can bind any p85 isoform with no apparent preference (see among others, refs. 3 and 4). p85s have a dual effect on the p110 subunits because they stabilize the thermally labile p110s but also conformationally inhibit their catalytic activity (5). Upon cellular stimulation, SH2 domain-mediated recruitment of p85/p110 complexes to Tyr phosphorylated (pY) membraneproximal proteins serves dual functions: It positions p110 in proximity with its substrates, and the engagement of the p85 SH2 domains relieves p85-mediated inhibition of p110, thus increasing enzymatic activity of p110 (6, 7).Experiments using gene-targeted mice and p110 isoformselective inhibitors have uncovered nonredundant physiological functions of the p110 isoforms. These functions include insulin signaling in metabolic tissues (p110␣; refs. 8 and 9), integrin signaling in platelets (p110␤; ref. 10) and signaling through a variety of receptors in leukocytes (p110␦; refs. 11-15). The mechanisms of this nonredundant signaling are not fully understood. Indeed, p110 isoforms have high homology in their primary sequence, interact nonselectively with the different p85s, and have the same lipid...

The class IA isoforms of phosphoinositide 3-kinase (p110α, p110β and p110δ) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110δ selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110δ in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110α, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110δ. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110α also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110δ.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110δ isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110δ-selective PI3K inhibitors may be more effective in inflammation than in cancer.

BackgroundDespite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110δ catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy.Principal FindingsWe show that p110δ expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110δ gene transcription appears to be constitutive and not subject to acute stimulation. 5′RACE experiments revealed that p110δ mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110δ transcripts are higher in leukocytes compared to non-leukocytes, with the p110δ transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110δ gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a.ConclusionThis study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110δ.