Uncovering the PI3Ksome: Phosphoinositide 3-Kinases and Counteracting PTEN Form a Signaling Complex with Intrinsic Regulatory Properties

Production of the phosphoinositide lipid phosphatidylinositol (3,4,5)trisphosphate [PI(3,4,5)P₃, or PIP₃] by class I phosphoinositide 3-kinases (PI3Ks) is a major signaling mechanism whose deregulation contributes to serious diseases, including cancer. New findings suggest that tyrosine kinase receptor engagement results in the assembly of hetero-oligomeric PI3K complexes in which PI3Kα first activates PI3Kβ, and PI3K catalytic activity then promotes recruitment and activation of the PIP₃-removing tumor suppressor PTEN. Thus, PIP₃ production is fine-tuned through formation of an intrinsically regulated “PI3Ksome.”     

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway

In chemotaxing ameboid cells, a complex leading-edge signaling circuit forms on the cytoplasmic leaflet of the plasma membrane and directs both actin and membrane remodeling to propel the leading edge up an attractant gradient. This leading-edge circuit includes a putative amplification module in which Ca²⁺-protein kinase C (Ca²⁺-PKC) is hypothesized to phosphorylate myristoylated alanine-rich C kinase substrate (MARCKS) and release phosphatidylinositol-4,5-bisphosphate (PIP₂), thereby stimulating production of the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃) by the lipid kinase phosphoinositide-3-kinase (PI3K). We investigated this hypothesized Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ amplification module and tested its key predictions using single-molecule fluorescence to measure the surface densities and activities of its protein components. Our findings demonstrate that together Ca²⁺-PKC and the PIP₂-binding peptide of MARCKS modulate the level of free PIP₂, which serves as both a docking target and substrate lipid for PI3K. In the off state of the amplification module, the MARCKS peptide sequesters PIP₂ and thereby inhibits PI3K binding to the membrane. In the on state, Ca²⁺-PKC phosphorylation of the MARCKS peptide reverses the PIP₂ sequestration, thereby releasing multiple PIP₂ molecules that recruit multiple active PI3K molecules to the membrane surface. These findings 1) show that the Ca²⁺-PKC-MARCKS-PIP₂-PI3K-PIP₃ system functions as an activation module in vitro, 2) reveal the molecular mechanism of activation, 3) are consistent with available in vivo data, and 4) yield additional predictions that are testable in live cells. More broadly, the Ca²⁺-PKC-stimulated release of free PIP₂ may well regulate the membrane association of other PIP₂-binding proteins, and the findings illustrate the power of single-molecule analysis to elucidate key dynamic and mechanistic features of multiprotein signaling pathways on membrane surfaces.     

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP₂ following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP₂ mediated by Nir2. Nir2 detects PIP₂ hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP₂ homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP₂ and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.     

Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation

Phosphoinositides are bioactive lipids essential in the regulation of cell signaling as well as cytoskeleton and membrane dynamics. Their metabolism is highly active in blood platelets where they play a critical role during activation, at least through two well identified pathways involving phospholipase C and phosphoinositide 3-kinases (PI3K). Here, using a sensitive high-performance liquid chromatography-mass spectrometry method recently developed, we monitored for the first time the profiling of phosphatidylinositol (PI), PIP, PIP₂ and PIP₃ molecular species (fatty-acyl profiles) in human and mouse platelets during the course of stimulation by thrombin and collagen-related peptide. Furthermore, using class IA PI3K p110α or p110β deficient mouse platelets and a pharmacological inhibitor, we show the crucial role of p110β and the more subtle role of p110α in the production of PIP₃ molecular species following stimulation. This comprehensive platelet phosphoinositides profiling provides important resources for future studies and reveals new information on phosphoinositides biology, similarities and differences in mouse and human platelets and unexpected dramatic increase in low-abundance molecular species of PIP₂ during stimulation, opening new perspectives in phosphoinositide signaling in platelets.     

PI(3,4,5)P3 potentiates phospholipase C-β activity

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP₃. In this study, PIP₃ was found to potentiate Ca²⁺-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP₃ accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP₃ accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP₃ in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.     

An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor-stimulated PC12 cells

The local accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP₃) and resulting activation of Rac1/Cdc42 play a critical role in nerve growth factor (NGF)–induced neurite outgrowth. To further explore the mechanism, we visualized PIP₃, phosphatidylinositol (3,4) bisphosphate, and Rac1/Cdc42 activities by fluorescence resonance energy transfer (FRET) imaging in NGF-stimulated PC12 cells. Based on the obtained FRET images, and with the help of in silico kinetic reaction model, we predicted that PI-5-phosphatase negatively regulates PIP₃ upon NGF stimulation. In agreement with this model, depletion of Src homology 2 domain–containing inositol polyphosphate 5-phosphatase 2 (SHIP2) markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP₃ accumulation and considerably increased the number and the length of neurites in phosphate and tensin homologue–depleted PC12 cells. Further refinement of the computational model predicted Rac1 regulation of PI3-kinase and SHIP2, which was also validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP₃ coordinately works with the PI3-kinase–mediated positive feedback to form an initial protrusive pattern and, later, to punctuate the PIP₃ accumulation to maintain proper neurite outgrowth.     

Phosphoinositides alter lipid bilayer properties

Phosphatidylinositol-4,5-bisphosphate (PIP₂), which constitutes ∼1% of the plasma membrane phospholipid, plays a key role in membrane-delimited signaling. PIP₂ regulates structurally and functionally diverse membrane proteins, including voltage- and ligand-gated ion channels, inwardly rectifying ion channels, transporters, and receptors. In some cases, the regulation is known to involve specific lipid–protein interactions, but the mechanisms by which PIP₂ regulates many of its various targets remain to be fully elucidated. Because many PIP₂ targets are membrane-spanning proteins, we explored whether the phosphoinositides might alter bilayer physical properties such as curvature and elasticity, which would alter the equilibrium between membrane protein conformational states—and thereby protein function. Taking advantage of the gramicidin A (gA) channels’ sensitivity to changes in lipid bilayer properties, we used gA-based fluorescence quenching and single-channel assays to examine the effects of long-chain PIP₂s (brain PIP₂, which is predominantly 1-stearyl-2-arachidonyl-PIP₂, and dioleoyl-PIP₂) on bilayer properties. When premixed with dioleoyl-phosphocholine at 2 mol %, both long-chain PIP₂s produced similar changes in gA channel function (bilayer properties); when applied through the aqueous solution, however, brain PIP₂ was a more potent modifier than dioleoyl-PIP₂. Given the widespread use of short-chain dioctanoyl-phosphoinositides, we also examined the effects of diC₈-phosphoinositol (PI), PI(4,5)P₂, PI(3,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. The diC₈ phosphoinositides, except for PI(3,5)P₂, altered bilayer properties with potencies that decreased with increasing head group charge. Nonphosphoinositide diC₈ phospholipids generally were more potent bilayer modifiers than the polyphosphoinositides. These results show that physiological increases or decreases in plasma membrane PIP₂ levels, as a result of activation of PI kinases or phosphatases, are likely to alter lipid bilayer properties, in addition to any other effects they may have. The results further show that exogenous PIP₂, as well as structural analogues that differ in acyl chain length or phosphorylation state, alters lipid bilayer properties at the concentrations used in many cell physiological experiments.     

The structural basis of Akt PH domain interaction with calmodulin

Akt plays a key role in the Ras/PI3K/Akt/mTOR signaling pathway. In breast cancer, Akt translocation to the plasma membrane is enabled by the interaction of its pleckstrin homology domain (PHD) with calmodulin (CaM). At the membrane, the conformational change promoted by PIP₃ releases CaM and facilitates Thr308 and Ser473 phosphorylation and activation. Here, using modeling and molecular dynamics simulations, we aim to figure out how CaM interacts with Akt’s PHD at the atomic level. Our simulations show that CaM-PHD interaction is thermodynamically stable and involves a β-strand rather than an α-helix, in agreement with NMR data, and that electrostatic and hydrophobic interactions are critical. The PHD interacts with CaM lobes; however, multiple modes are possible. IP₄, the polar head of PIP₃, weakens the CaM-PHD interaction, implicating the release mechanism at the plasma membrane. Recently, we unraveled the mechanism of PI3Kα activation at the atomistic level and the structural basis for Ras role in the activation. Here, our atomistic structural data clarify the mechanism of how CaM interacts, delivers, and releases Akt—the next node in the Ras/PI3K pathway—at the plasma membrane.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Modulation of the Olfactory CNG Channel by Ptdlns(3,4,5)P<Subscript>3</Subscript>

Recent data suggest that the 3-phosphoinositides can modulate cyclic nucleotide signaling in rat olfactory receptor neurons (ORNs). Given the ability of diverse lipids to modulate ion channels, we asked whether phosphatidylinositol 3,4,5-trisphosphate (PIP₃) can regulate the olfactory cyclic nucleotide-gated (CNG) channel as a possible mechanism for this modulation. We show that applying PIP₃ to the intracellular side of inside-out patches from rat ORNs inhibits activation of the olfactory CNG channel by cAMP. The effect of PIP₃ is immediate and partially reversible, and reflects an increase in the EC₅₀ of cAMP, not a reduction in the single-channel current amplitude. The effect of PIP₃ is significantly stronger than that of PIP₂; other phospholipids tested have no appreciable effect on channel activity. PIP₃ similarly inhibits the recombinant heteromeric (A2/A4) and homomeric (A2) olfactory CNG channel expressed in HEK293 cells, suggesting that PIP₃ acts directly on the channel. These findings indicate that 3-phosphoinositides can be functionally important regulators of CNG channels.     

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40–75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP₂) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP₂ inhibited the enzyme (16%). The inhibition of the protease by PIP₂ was concentration-dependent. During receptor-coupled cell activation, phospholipase A₂ (PLA₂) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP₂ by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP₂ to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA₂, PI 4-kinase, PIP 5-kinase and PLC are involved.     

Stimulation of phosphoinositide degradation and phosphatidylinositol-4-phosphate phosphorylation by GTP exclusively in plasma membrane of rat brain

The effect of GTP on the hydrolysis of [³H]phosphatidyinositol (PI), [³H]phosphatidylinositol-4-phosphate (PIP) and [³H]phosphatidylinositol-4,5-bisphosphate (PIP₂) by phospholipase C of rat brain plasma membrane, microsomes and cytosol was determined. Moreover the regulation of PI and PIP phosphorylation by GTP in brain plasma membrane was investigated.In the presence of EGTA PIP₂ was actively degradted, opposite to PI and PIP which require Ca²⁺ for their hydrolysis. Addition of calcium ions in each case caused stimulation of inositide phosphodiesterase(s). GTP independently of calcium ions activates by about 3 times phospholipase C acting on PIP and PIP₂ exclusively in the plasma membrane. PI degradation was unaffected by GTP. In the presence of Ca²⁺ guanine nucleotides have synergistic stimulatory effect on plasma membrane bound phospholipase C acting on PIP₂. PIP kinase of brain plasma membrane was stimulated by GTP by about 20–100% in the presence of exogenous and endogenous substrate respectively. PI kinase was negligible activated by about 20% exclusively in the presence of endogenous substrate. These results indicated that guanine nucleotide modulates the level of second messengers as diacylglycerol and IP₃ through the activation of phospholipase C acting on PIP₂ exclusively in brain plasma membrane. The stimulation of phospholipase C by GTP may occur directly or through the enhancement of substrate level PIP₂ due to stimulation of PIP kinase.     

New Functions for PI3K in the Control of Cell Division

Although cell lipids were initially envisioned as structural components of the cell, their essential contribution to initiation and regulation of cell responses is now clearly established. Among the different lipids that regulate cell responses, those produced by class I phosphoinositide 3-kinase (PI3K), phosphatidylinositol (3,4)P₂ (PIP₂), and phosphatidylinositol (3,4,5)P₃ (PIP₃), have concentrated much attention in recent years. PIP2 and PIP3 are involved in cell division and survival control, and mutations in the PI3K pathway are linked to autoimmunity and cancer. Here we discuss two novel observations: a PI3K function in the late-G₁ phase of the cell cycle and the contribution of the p85 PI3K regulatory subunit in the control of cytokinesis.     

Probing the Architecture,Dynamics, and Inhibition of the PI4KIIIα/TTC7/FAM126 Complex

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα) is the lipid kinase primarily responsible for generating the lipid phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, which acts as the substrate for generation of the signaling lipids PIP₂ and PIP₃. PI4KIIIα forms a large heterotrimeric complex with two regulatory partners, TTC7 and FAM126. We describe using an integrated electron microscopy and hydrogen–deuterium exchange mass spectrometry (HDX-MS) approach to probe the architecture and dynamics of the complex of PI4KIIIα/TTC7/FAM126. HDX-MS reveals that the majority of the PI4KIIIα sequence was protected from exchange in short deuterium pulse experiments, suggesting presence of secondary structure, even in putative unstructured regions. Negative stain electron microscopy reveals the shape and architecture of the full-length complex, revealing an overall dimer of PI4KIIIα/TTC7/FAM126 trimers. HDX-MS reveals conformational changes in the TTC7/FAM126 complex upon binding PI4KIIIα, including both at the direct TTC7-PI4KIIIα interface and at the putative membrane binding surface. Finally, HDX-MS experiments of PI4KIIIα bound to the highly potent and selective inhibitor GSK-A1 compared to that bound to the non-specific inhibitor PIK93 revealed substantial conformational changes throughout an extended region of the kinase domain. Many of these changes were distant from the putative inhibitor binding site, showing a large degree of allosteric conformational changes that occur upon inhibitor binding. Overall, our results reveal novel insight into the regulation of PI4KIIIα by its regulatory proteins TTC7/FAM126, as well as additional dynamic information on how selective inhibition of PI4KIIIα is achieved.     

Mal connects TLR2 to PI3Kinase activation and phagocyte polarization

The recognition of bacterial lipoproteins by toll‐like receptor (TLR) 2 is pivotal for inflammation initiation and control in many bacterial infections. TLR2‐dependent signalling is currently believed to essentially require both adaptor proteins MyD88 (m yeloid d ifferentiation primary response gene 88) and Mal/TIRAP (M yD88‐a dapter‐l ike/TI R‐domain‐containing a daptor p rotein). TLR2‐dependent, but MyD88‐independent responses have not been described yet. We report here on a novel‐signalling pathway downstream of TLR2, which does not adhere to the established model. On stimulation of the TLR2/6 heterodimer with diacylated bacterial lipoproteins, Mal directly interacts with the regulatory subunit of phosphoinositide 3‐kinase (PI3K), p85α, in an inducible fashion. The Mal–p85α interaction drives PI3K‐dependent phosphorylation of Akt, phosphatidylinositol(3,4,5)P3 (PIP₃) generation and macrophage polarization. MyD88 is not essential for PI3K activation and Akt phosphorylation; however, cooperates with Mal for PIP₃ formation and accumulation at the leading edge. In contrast to TLR2/6, TLR2/1 does not require Mal or MyD88 for Akt phosphorylation. Hence, Mal specifically connects TLR2/6 to PI3K activation, PIP₃ generation and macrophage polarization.     

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP₂ is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP₂ in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP₂ increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP₂. In contrast, PIP5Kα knockdown that reduced PIP₂ levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP₂-dependent recruitment of TIRAP to the plasma membrane.     

PIP3-dependent macropinocytosis is incompatible with chemotaxis

In eukaryotic chemotaxis, the mechanisms connecting external signals to the motile apparatus remain unclear. The role of the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP₃) has been particularly controversial. PIP₃ has many cellular roles, notably in growth control and macropinocytosis as well as cell motility. Here we show that PIP₃ is not only unnecessary for Dictyostelium discoideum to migrate toward folate, but actively inhibits chemotaxis. We find that macropinosomes, but not pseudopods, in growing cells are dependent on PIP₃. PIP₃ patches in these cells show no directional bias, and overall only PIP₃-free pseudopods orient up-gradient. The pseudopod driver suppressor of cAR mutations (SCAR)/WASP and verprolin homologue (WAVE) is not recruited to the center of PIP₃ patches, just the edges, where it causes macropinosome formation. Wild-type cells, unlike the widely used axenic mutants, show little macropinocytosis and few large PIP₃ patches, but migrate more efficiently toward folate. Tellingly, folate chemotaxis in axenic cells is rescued by knocking out phosphatidylinositide 3-kinases (PI 3-kinases). Thus PIP₃ promotes macropinocytosis and interferes with pseudopod orientation during chemotaxis of growing cells.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

Gα16 interacts with Class IA phosphatidylinositol 3-kinases and inhibits Akt signaling

Phosphatidylinositol 3-kinase (PI3K) mediates receptor tyrosine kinase and G protein coupled receptor (GPCR) signaling by phosphorylating phosphoinositides to elicit various biological responses. Gα_q has previously been shown to inhibit class IA PI3K by interacting with the p110α subunit. However, it is not known if PI3Ks can associate with other Gα_q family members such as Gα₁₆. Here, we demonstrated that class IA PI3Ks, p85/p110α and p85/p110β, could form stable complexes with wild type Gα₁₆ and its constitutively active mutant (Gα₁₆QL) in HEK293 cells. In contrast, no interaction between Gα₁₆ and class IB PI3K was observed. The Gα₁₆/p110α signaling complex could be detected in hematopoietic cells that endogenously express Gα₁₆. Overexpression of class I PI3Ks did not inhibit Gα₁₆QL-induced IP₃ production and, unlike p63RhoGEF, class IA PI3Ks did not attenuate the binding of PLCβ₂ to Gα₁₆QL. On the contrary, the function of class IA PI3Ks was suppressed by Gα₁₆QL as revealed by diminished production of PIP₃ as well as inhibition of EGF-induced Akt phosphorylation. Taken together, these results suggest that Gα₁₆ can bind to class IA PI3Ks and inhibit the PI3K signaling pathway.