Abstract

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P₃, the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses^{1, 2}. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gs) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gs, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)K, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P₃, activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)K to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gs, p101 and the Ras–RBD interaction all have important roles in the regulation of PI(3)K in vivo and that they can simultaneously, but differentially, control distinct PI(3)K effectors.

Introduction

Class I PI(3)Ks are heterodimers made up of regulatory and p110 catalytic subunits. The mammalian class I PI(3)Ks are divided into two subfamilies: the class IA PI(3)Ks (named after their catalytic subunits, p110, and ), which are associated with SH2 domain-containing adaptor subunits and can be activated by tyrosine kinase-based mechanisms; and the class IB PI(3)K (p110), which associates with either p84 (ref. 3; also called p87^PIKAP, ref. 4) or p101 adaptors⁵, through which it can be activated by Gs. All class I PI(3)K catalytic subunits can bind, and be activated by, GTP-Ras proteins^{6, 7} (with the possible exception of PI(3)K).

PI(3)K is most highly expressed in neutrophils, where it is predominantly associated with p101 (approximately 95%)³. Biochemical and cellular studies have shown that p101/p110 is substantially activated by Gs and in a manner that is dependent on p101 (refs 5, 9–11). Recombinant PI(3)K has also been shown to be activated by GTP-Ras proteins (N, K, H and R) directly and independently of other regulators (such as Gs or phosphotyrosine residues^{7, 12}). The crystal structures of p110, both free and bound to active Ras, have shown that five residues in p110 are critical for binding GTP-Ras; substitutions at these sites create a mutant protein (DASAA; T232D, K251A, K254S, K255A and K256A) that can neither bind or be activated by GTP-Ras in vitro or in transfected cells⁷.

Neutrophils are circulating, myeloid-derived cells. Adjacent to sites of injury or infection, neutrophils rapidly exit the circulation and migrate through the surrounding basement membrane towards ongoing damage¹³. This migration process is underpinned by the chemotactic ability of neutrophils to move towards sources of a variety of inflammatory mediators (for example, fMLP, IL-8 MIP-2 and Kc, the murine analogue of Gro-). As neutrophils approach sites of inflammation they become primed through exposure to a number of cytokines released in this context, such as lipopolysaccharide (LPS) and tumour necrosis factor (TNF). This sensitises the neutrophils and dramatically increases their ability to both phagocytose apoptotic cells or pathogens and release ROS that are involved in killing¹⁴. These functional responses enable neutrophils to drive the resolution of inflammatory responses.

Mice survive genetic ablation of PI(3)K; however, PI(3)K- deficient neutrophils fail to respond appropriately to various inflammatory mediators^{15, 16, 17}. Accumulation of PtdIns(3,4,5)P₃, phosphorylation of PKB, production of ROS and chemotaxis in response to ligands such as fMLP and C5a are all severely inhibited. Use of isoform-selective PI(3)K inhibitors has shown that PI(3)K can act as a master kinase that controls subsequent activation of PI(3)Ks and ¹⁸. Detailed study of PI(3)K- deficient mice has shown that this protein has important functions in the heart, lymphocytes, endothelium, platelets and mast cells, indicating the broad significance of these signalling cascades^{19, 20, 21}.

The accepted dogma is that PI(3)K is rapidly activated by Gs that are released by dissociation of G_i-containing G-proteins, because PtdIns(3,4,5)P₃ signals in these responses peak within 12 s and typically decline within 1 min^{22, 23, 24}. The role of Ras is likely to be relatively minor because of these rapid kinetics, but little work has yet addressed the role of Ras in regulation of PI(3)Ks in vivo. We have asked whether there is a role for endogenous Ras in regulating PI(3)K in mouse neutrophils by creating a PI3K^DASAA knock-in.

Published work in the field is consistent with the hypothesis that p101 is needed for G activation of PI(3)K, that it only functions in a complex with PI(3)K and that it is not essential for Ras activation of p110^{5, 7, 11}. These observations suggest loss of p101 would serve as a specific means of assessing the role of Gs, as distinct from Ras, in the activation of PI(3)K. We have created mice lacking p101 in an attempt to address these issues.

An important question in PI(3)K signalling, and other signalling networks transducing many distinct receptor signals, is how is specificity delivered? There is increasing evidence that, despite the similarities between the class I PI(3)Ks both in terms of regulatory properties and lipid product, they can be differentially coupled to distinct effectors and cellular responses in single cell types, probably through subtle differences in their biochemical properties. The potential for differential regulation of PI(3)K effectors through a single species of PI(3)K in a single cell type has not been as closely considered.

Hence, the combined analysis of p101^-/- and PI(3)K^DASAA/DASAA mice also enables a currently unique window into the relative regulatory power of adaptor subunit-mediated versus Ras-mediated signals on class I PI(3)K activity and, furthermore, the potential for an analysis of the ability of a single PI(3)K to differentially control effectors as a function of its mode of activation.

The mouse p101 locus was disrupted in E14 stem cells by the in-frame insertion of a -Gal gene at codon 11. p101^-/- mice were viable, fertile, of normal size and with normal blood counts (Methods and see Supplementary Information, Fig. S1). Western blotting showed that neutrophils from p101^-/- mice contained no detectable p101 and unchanged expression of p110, , or (Fig. 1). Significantly, because the p84 adaptor for PI(3)K is the neighbouring gene to p101 on mouse chromosome 11, expression of p84 message and protein was not reduced; in fact, it was increased by approximately 50% compared with wild type (this should be viewed in the context that approximately 95% of p110 in mouse neutrophils is bound to p101 and 5% to p84).

Figure 1: Expression and distributions of PI(3)K subunits in neutrophils isolated from p101^-/- and p110^DASAA/DASAA mice.

(a) Western immunoblots demonstrating the loss in expression of p101, unchanged expression of PI(3)K catalytic subunits, appearance of -galactosidase and increased expression of p84 in neutrophils from p101^-/- mice, compared with p101^+/+ mice. (b) Quantification of immunoblots (similar to a) comparing expression of PI(3)K subunits in neutrophils from p101^-/- compared with p101^+/+ mice. Endogenous GAPDH was quantified in parallel to demonstrate equivalent loading in experiments analysing p110 expression. Data are means s.e.m. (n values are as indicated). (c) Expression of PI(3)K in neutrophils from p110^+/+, p110^+/- and p110^DASAA/DASAA (D/D) mice. Western immunoblots were probed in parallel with anti-p110 and anti-p67^phox antibodies and the ratio of p110:p67^phox expression was quantified for each individual sample and compared with those from p110^+/+ mice (scaled as 1.0). Very similar ratios were obtained if p110 expression was normalised through -COP (coat-protein complex; data not shown). Data are means s.e.m. (n = 3, from 3 independent experiments). (d) Distribution of p110 in neutrophils from p101^-/-, p110^DASAA/DASAA and wild-type mice. Neutrophils were sonicated to prepare an homogenate (H) and ultracentrifuged to derive cytosolic (C) and membrane fractions (M), which were immunoblotted (C and H, 3.5 10⁵; and M, 3.5 10⁶ cell equivalents per lane, respectively) to quantify p110, G and GAPDH in each of the fractions relative to the starting homogenate (100%). Data are from two experiments and are means s.e.m. (n = 4; see also Supplementary Information, Fig. S3).

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A p110^DASAA (p110^DASAA/DASAA) knock-in mouse was created using standard approaches (Methods and see Supplementary Information, Fig. S2). Pure, recombinant 6His–p110^DASAA has the same basal catalytic activity, ability to bind p101 and activation by Gs as 6His–p110, prepared in parallel (see Supplementary Information, Fig. S3). In the context of published data⁷ this suggests that p110^DASAA should be bound by p101 (or p84), and be fully sensitive to Gs but insensitive to Ras. p110^DASAA/DASAA mice were viable, fertile, of normal size and with generally normal blood counts (see Supplementary Information, Fig. S1), and quantitative western blotting indicated that expression of other class I PI(3)K catalytic subunits was normal (data not shown), but expression of p110^DASAA/DASAA was reduced (Fig. 1). This reduction in expression of p110^DASAA/DASAA could be because of minor changes in the stability of the p110^DASAA/DASAA protein that are secondary to the amino-acid substitutions, or the levels of p110 message, caused by the small quantity of foreign, intronic sequence remaining after the gene targeting. To control for any potentially confounding effects that these small reductions in PI(3)K-subunit expression may have had on our analysis of p110^DASAA/DASAA mice, we also compared them with p110^+/- mice.

Analysis of the subcellular distribution of endogenous p110 in p101^-/- or p110^DASAA/DASAA-expressing versus wild-type neutrophils showed, however, that these genetic modifications or minor changes in expression had no effect on the ability of PI(3)K to associate with cell membranes in vivo (Fig. 1 and see Supplementary Information, Fig. S3).

A variety of cell-based assays were used to assess the overall integrity of G-protein dependent signalling in neutrophils isolated from p101^-/- and p110^DASAA/DASAA mice. fMLP and C5a-stimulated phosphorylation of p42/44 ERKs, p38 and JNK with the same dose-dependency and maximal effects as in wild-type cells, and activation of Ras proteins by fMLP, as assessed by Raf–RBD pulldown, was also unchanged in both genetically manipulated lines (see Supplementary Information, Fig. S4).

To establish the degree to which PI(3)K could be activated in response to G-protein-coupled agonists in neutrophils from p101^-/- and p110^DASAA/DASAA mice, the accumulation of its catalytic products, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, was measured. Substantial reductions in the accumulation of these two lipids was found in response to both fMLP and C5a, in neutrophils from both the p101^-/- and p110^DASAA/DASAA mice (Fig. 2 and data not shown). The reductions in PtdIns(3,4,5)P₃ accumulation in neutrophils from p101^-/- mice were particularly marked at sub-maximal doses of chemoattractant (Fig. 2e). The decrease in PtdIns(3,4,5)P₃ accumulation in response to fMLP observed in neutrophils from p110^-/- mice and reported by our group¹⁸ and others^{15, 16, 17}, was only slightly greater than those we report here (approximately 90% and 75% reductions at 10 s and 1 min, respectively).

Figure 2: Accumulation of PtdIns(3,4,5)P₃ is reduced in neutrophils isolated from p110^DASAA/DASAA (p110^D/D) and p101^-/- mice.

(a–e) ³²P-Pi labelled neutrophils from either p101^+/+ (solid line) versus p101^-/- (short-dashed line) mice (a, c, e) and p110^+/+ (solid line) versus p110^DASAA/DASAA (long-dashed line) mice (b, d) were stimulated for various times with fMLP (10 M; a and b) or C5a (100 nM; c and d), or for 10 s with various doses of C5a (e). PtdIns(3,4,5)P₃ (and other phosphoinositide) levels were quantified by deacylation, HPLC and scintillation counting. Data presented are one experiment respresentative of four in a; means s.e.m. (n = 3, except 60 s p110^DASAA/DASAA, which was a range of n = 2) in b; means range (n = 2, except p101^-/- 10 s and p101^+/+ 2 min, which were n = 1) in c; means s.e.m. (n = 3, except p110^DASAA/DASAA 1 min, which was a range of n = 2) in d; and means range (n = 2, except 0.6 nM C5a, which were s.e.m., n = 3) in e.

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In keeping with the reduced accumulation of PtdIns(3,4,5)P₃ and PtdIns(3,4,)P₂, activation of PKB, as measured by the phosphorylation status of Ser 473 in response to fMLP and C5a, was reduced in neutrophils from both p101^-/- and p110^DASAA/DASAA but not p110^+/- mice (Fig. 3).

Figure 3: Phosphorylation of Ser 473-PKB in response to chemoattractants is reduced in neutrophils from p101^-/- and p110^DASAA/DASAA mice.

(a) Immunoblots showing that phosphorylation of Ser 473-PKB, in response to various doses of C5a, is reduced in neutrophils derived from p101^-/- mice. (b) Quantification of data from experiments similar to that shown in a, Data presented are means s.e.m. (n = 4, from four experiments). (c) Quantification of immunoblots comparing the phosphorylation of Ser 473-PKB, in response to 10 M fMLP, in neutrophils from p110^+/+, p110^+/- and p110^DASAA/DASAA (p110^D/D) mice. Data are means s.e.m. (n = 3, p110^+/-; n = 4, p110^+/+; n = 4, p110^DASAA/DASAA).

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The ability of neutrophils from p110^-/-, p101^-/-, p110^+/-, p110^DASAA/DASAA and wild-type control mice to respond chemotactically or chemokinetically to fMLP, C5a, Kc and MIP2 was assessed. The chemotactic and chemokinetic responses of neutrophils from p110^-/- (as reported previously^{15, 16, 17, 25}), p101^-/- and p110^DASAA/DASAA, but not p110^+/- mice, were significantly and similarly reduced for all four inflammatory mediators (Fig. 4). Furthermore, in a standard in vivo model (thioglycollate-induced aseptic peritonitis) neutrophils from both p101^-/- and p110^DASAA/DASAA mice, similarly to those from p110^-/- mice^{15, 16, 17}, exhibited substantially reduced recruitment into the peritoneum (Fig. 4).

Figure 4: The migration of neutrophils from mice lacking p110 or p101 or expressing p110^DASAA/DASAA is substantially reduced.

(a) Cells (1 10⁶) from mice of the indicated genotype were loaded into the upper chamber of a transwell insert. In gradient mode, the chemoattractants were only placed in the lower chamber (MIP-2, 1 nM; Kc, 3 nM; fMLP, 1 M; C5a, 2 nM; all sub-maximal concentrations in this assay). us, unstimulated. Migration was assessed by counting the neutrophils that had appeared in the lower chamber after 1 h. Data are presented as percentage of wild-type cells that had migrated in gradient mode towards MIP-2 (called 100%). Means s.e.m. (n values are as indicated). (b) The migration of neutrophils in the presence of uniform concentrations of chemoattractant was measured, as described in a. Results are presented as percentage of migration to MIP-2 measured in gradient mode shown in a. (c) The migration of neutrophils into an aseptically inflamed peritoneum depends on p101 and the RBD of p110. Thioglycollate was injected into the peritoneum of various mice (genotypes indicated). After 4.5 h the number of neutrophils in the peritoneum of both the injected and un-injected (ui) control mice was quantified. The data presented are means s.e.m. (n values are as indicated). The numbers of neutrophils in the un-injected controls of the various genotypes were not significantly different and hence were pooled for presentation.

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The ability of neutrophils from p101^-/-, p110^+/- and p110^DASAA/DASAA mice, to produce ROS in response to fMLP or C5a was assayed and compared with neutrophils from p110^-/- mice and wild-type controls. As anticipated, there was a substantial and equivalent reduction in ROS generation by both primed and unprimed neutrophils from p110^DASAA/DASAA and p110^-/- mice, compared with controls. Surprisingly, there was no significant difference in ROS formation between neutrophils from p101^-/- and p101^+/+ mice, in response to a wide range of sub-maximal (in the context of our observation that PtdIns(3,4,5)P₃ production is most reduced in p101^-/- neutrophils at lower agonist concentrations) and maximal doses of fMLP and C5a, and in both primed and unprimed neutrophils (Fig. 5 and data not shown).

Figure 5:  Production of ROS in response to chemoattractants is reduced in neutrophils from p110^DASAA/DASAA but not p101^-/- mice.

(a) Production of ROS by unprimed mouse neutrophils is dependent on the RBD of p110 but not p101. Formation of ROS in response to fMLP, C5a and phorbol myristate acetate (PMA; 1 M, 100 nM and 300 nM final concentrations; which were all sub-maximal) was quantified by chemiluminescense assay. Data are presented as percentages of the response of neutrophils isolated from wild-type mice s.e.m. (n values are as indicated). (b) Production of ROS in response to fMLP by isolated, primed (TNF-) murine neutrophils (genotypes as indicated; the single, open-squared symbol shows parallel data obtained with neutrophils from p110^-/- mice), data are presented in relative response units as means s.e.m. (n = 8, p110^+/+; n = 6, p110^D/D; n = 3, p110^+/-;). Similar experiments with TNF- primed, p101^-/- neutrophils showed no defect in ROS accumulation in response to neither fMLP nor C5a (data not shown). (c) Production of ROS in response to sub-maximal stimulation with C5a in unprimed neutrophils from p101^+/+ (solid lines) and p101^-/- mice. Data are presented as percentage of maximum response of that genotype s.e.m. (n = 4, from one experiment typical of four). No p101-dependent defect in ROS accumulation is revealed at a range of sub-maximal concentrations of C5a. (d) Data as in c, except the cells were stimulated with sub-maximal concentrations of fMLP.

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These results demonstrate an essential role for p101 in signalling through PI(3)K to PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, activation of PKB and migration both in transwell assays and in vivo, indicating that the roles of p101 in mouse neutrophils overlap with those of p110. The extent of the roles for p101 specifically, and PI(3)K adaptors generally, in these PI(3)K-dependent responses are probably slightly underestimated through limited redundancy between p84 and p101 and the 50% increase in p84 expression in the absence of p101.

The above results and argument suggest the scale of the role of Gs in activating PI(3)K will be approximated by the loss of function resulting from p101 deficiency. Therefore, we conclude that our results also demonstrate that, as expected, Gs have substantial roles in the regulation of PI(3)K activity in mouse neutrophils.

Currently, the identity of the signalling pathway regulating the RBD of PI(3)K in these contexts is not clear. An extensive screen of the ability of Ras-related GTPases to regulate class I PI(3)Ks, in both transfected mammalian cells and in vitro, has found that a variety of GTP-bound Ras proteins (for example, N-, K-, R- and H-Ras) could activate PI(3)K, but Rap and other closely related proteins (Rap 1 and 2, Rheb or Rin) could not^{8, 12}. Therefore, it is very likely that a Ras family GTPase regulates this domain in vivo. A variety of G-protein-activating ligands can stimulate rapid and substantial increases in the amount of total GTP-Ras in both mouse and human neutrophils²⁸ (for example, 20–40-fold increase in 10 s; see Supplementary Information, Fig. S4). The species of Ras protein involved has not been characterised, but neutrophils probably contain K- and N-, but not H-, Ras. Similarly, the identity of the Ras-GEF (guanine nucleotide exchange factor), and how it is activated in these contexts, is uncertain, although a variety of results with various pharmacological inhibitors indicate no role for Src-type tyrosine kinases or phospholipase C activity²⁹.

Several further important conclusions can be drawn from a comparison of the phenotypes resulting from loss of p101 and knock-in of a Ras-insensitive form of PI(3)K. First, we can show that in a signalling cascade engaged by a single receptor, signals through both G–p101 and Ras–p110 are essential for normal activation. Second, a quantitative analysis of the scale of loss of PtdIns(3,4,5)P₃ accumulation in neutrophils from p101^-/- and p110^DASAA/DASAA mice, mapped against our (and others) work previously defining the reduction in PtdIns(3,4,5)P₃, suggests that Ras and G signals combine synergistically during physiological engagement of this pathway. This property is clearly translated into assays of migration in vivo and is entirely consistent with previous work indicating Ras and G can synergistically activate PI(3)K 'in transfecto'⁷. Third, different regulators of PI(3)Ks (here, Gs and Ras) can simultaneously but differentially regulate distinct PI(3)K effectors.Very similar extents of loss and kinetics of PtdIns(3,4,5)P₃ accumulation were observed in response to fMLP in neutrophils from p101^-/- and p110^DASAA/DASAA mice, making it difficult to explain the differential regulation of ROS formation on the basis of temporal specificity or intensity thresholds for responses. Furthermore, previous use of PI(3)K catalytic-site inhibitors would suggest that the reduction in PtdIns(3,4,5)P₃ production observed in p101^-/- neutrophils should be associated with significant reductions in ROS formation⁷. The likely explanation is that the Ras and p101 signals are spatially distinct and that this translates into differences in the sites of activation of PI(3)K and accumulation of PtdIns(3,4,5)P₃, and ultimately to qualitative differences in activation of effectors. This suggests that the class I PI(3)K cassette can use a combination of multiple PI(3)K isoforms and has the ability to decode different regulators independently providing flexibility and specificity to its signalling.

Note added in proof: a related manuscript byOrme et al. (Nature Cell Biol. 8, doi: 10.1038/ncb1493; 2006)is also published in this issue.

Acknowledgements

PI(3)K mice were provided by M. Wymann. Thanks to M. Skynner for help in the initial isolation of p101 genomic clones and thanks also to T. Green for use of a blood-cell counter. Thanks to P. Arnaud for design of PCR primers and A. Segonds-Pichon for help with statistics. C.E. was a Beit fellow. A.M.C. was supported by a Wellcome Trust Intermediate fellowship. Different parts of this work were funded by grants from the Biotechnology and Biological Sciences Research council (BBSRC) and Cancer Research UK (CRUK).

Competing interests statement

The authors declare no competing financial interests.

Received 26 July 2006; Accepted 21 August 2006; Published online 15 October 2006.

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