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G protein sensitive phosphoinositide 3-kinase gamma (PI3Kgamma) has been characterised as a pleiotropic signalling protein expressing lipid kinase and protein kinase activities. Whereas the regulation of the lipid kinase activity has been investigated in detail, the regulatory features of PI3Kgamma protein kinase activity are unknown. Here we report that Gbetagamma subunits of heterotrimeric G proteins induce a biphasic response of PI3Kgamma autophosphorylation in vitro, which contrasts the regulatory effects of the G proteins on PI3Kgamma lipid kinase activity. In addition to autophosphorylation PI3Kgamma is able to catalyse transphosphorylation of the adapter protein p101 and the protein kinase MEK-1. In the presence of the p101, Gbetagamma affects PI3Kgamma protein kinase activities in a complex manner. In summary, the differential regulatory effects of heterotrimeric G proteins on PI3Kgamma lipid and protein kinase activities in vitro reflect the functional diversity of the enzyme observed in vivo.

Colony-stimulating factor 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2. A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities of PI-3 kinase in these complexes support the notion that the activity of PI-3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.

Signaling by phosphorylated species of phosphatidylinositol (PI) appears to regulate diverse responses in eukaryotic cells. A differential display screen for fat- and muscle-specific transcripts led to identification and cloning of the full-length cDNA of a novel mammalian 2,052-amino-acid protein (p235) from a mouse adipocyte cDNA library. Analysis of the deduced amino acid sequence revealed that p235 contains an N-terminal zinc-binding FYVE finger, a chaperonin-like region in the middle of the molecule, and a consensus for phosphoinositide 5-kinases at the C terminus. p235 mRNA appears as a 9-kb transcript, enriched in insulin-sensitive cells and tissues, likely transcribed from a single-copy gene in at least two close-in-size splice variants. Specific antibodies against mouse p235 were raised, and both the endogenously and heterologously expressed proteins were biochemically detected in 3T3-L1 adipocytes and transfected COS cells, respectively. Immunofluorescence microscopy analysis of endogenous p235 localization in 3T3-L1 adipocytes with affinity-purified anti-p235 antibodies documented a punctate peripheral pattern. In COS cells, the expressed p235 N-terminal but not the C-terminal region displayed a vesicular pattern similar to that in 3T3-L1 adipocytes that became diffuse upon Zn2+ chelation or FYVE finger truncation. A recombinant protein comprising the N-terminal but not the C-terminal region of the molecule was found to bind 2.2 mole equivalents of Zn2+. Determination of the lipid kinase activity in the p235 immunoprecipitates derived from 3T3-L1 adipocytes or from COS cells transiently expressing p235 revealed that p235 displayed unique preferences for PI substrate over already phosphorylated PI. In conclusion, the mouse p235 protein determines an important novel class of phosphoinositide kinases that seems to be targeted to specific intracellular loci by a Zn-dependent mechanism.

Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32PO4-labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.

HLA-specific killer cell inhibitory receptors (KIR) are thought to impede natural killer (NK) and T cell activation programs through recruitment of the SH2 domain-containing tyrosine phosphatases, SHP-1 and SHP-2, to their cytoplasmic tails (CYT). To identify other SH2 domain-containing proteins that bind KIR CYT, we used the recently described yeast two-bait interaction trap and a modified version of this system, both of which permit tyrosine phosphorylation of bait proteins. Using these systems, we show that KIR CYT, once phosphorylated by the src-family tyrosine kinase LCK, additionally bind the p85α regulatory subunit of phosphatidylinositol (PI) 3-kinase. Furthermore, we show that in an NK cell line, NK3.3, cross-linking of KIR results in recruitment of p85α to KIR and activation of PI 3-kinase lipid kinase activity. One consequence of KIR coupling to PI 3-kinase is downstream activation of the antiapoptotic protein kinase AKT. Therefore, in addition to providing negative signals, KIR may also contribute positive signals for NK and T cell growth and/or survival.

The cellular effects of MCP-1 are mediated primarily by binding to CC chemokine receptor-2. We report here that MCP-1 stimulates the formation of the lipid products of phosphatidylinositol (PI) 3-kinase, namely phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PI 3,4,5-P3) in THP-1 cells that can be inhibited by pertussis toxin but not wortmannin. MCP-1 also stimulates an increase in the in vitro lipid kinase activity present in immunoprecipitates of the class 1A p85/p110 heterodimeric PI 3-kinase, although the kinetics of activation were much slower than observed for the accumulation of PI 3,4,5-P3. In addition, this in vitro lipid kinase activity was inhibited by wortmannin (IC50 = 4.47 +/- 1.88 nM, n = 4), and comparable concentrations of wortmannin also inhibited MCP-stimulated chemotaxis of THP-1 cells (IC50 = 11.8 +/- 4.2 nM, n = 4), indicating that p85/p110 PI 3-kinase activity is functionally relevant. MCP-1 also induced tyrosine phosphorylation of three proteins in these cells, and a fourth tyrosine-phosphorylated protein co-precipitates with the p85 subunit upon MCP-1 stimulation. In addition, MCP-1 stimulated lipid kinase activity present in immunoprecipitates of a class II PI 3-kinase (PI3K-C2alpha) with kinetics that closely resembled the accumulation of PI 3,4,5-P3. Moreover, this MCP-1-induced increase in PI3K-C2alpha activity was insensitive to wortmannin but was inhibited by pertussis toxin pretreatment. Since this mirrored the effects of these inhibitors on MCP-1-stimulated increases in D-3 phosphatidylinositol lipid accumulation in vivo, these results suggest that activation of PI3K-C2alpha rather than the p85/p110 heterodimer is responsible for mediating the in vivo formation of D-3 phosphatidylinositol lipids. These data demonstrate that MCP-1 stimulates protein tyrosine kinases as well as at least two separate PI 3-kinase isoforms, namely the p85/p110 PI 3-kinase and PI3K-C2alpha. This is the first demonstration that MCP-1 can stimulate PI 3-kinase activation and is also the first indication of an agonist-induced activation of the PI3K-C2alpha enzyme. These two events may play important roles in MCP-1-stimulated signal transduction and biological consequences.

Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

Peripheral blood cell DNA from a patient with a chronic myeloproliferative disorder was tested in the tumorigenicity assay. Upon tumor induction in nude mice we isolated a human oncogene by means of genomic cloning, exon trap analysis and cDNA cloning. Sequence analysis revealed a fusion product of the p85beta subunit of phosphatidylinositol (PI) 3-kinase and HUMORF8, a putative deubiquitinating enzyme, which has been generated during the DNA transfection process. Application of the tumorigenicity assay to various p85beta and HUMORF8 cDNA constructs indicated that the recombination of both genes rather than the truncation of one of the fusion partners renders the chimeric protein tumorigenic. Moreover, sequence analysis of human wildtype p85beta revealed an alanine for serine substitution at a site important for the regulation of the lipid kinase activity of PI 3-kinase in human p85alpha. This variation may relate to differences in the mode of signal transduction from both p85 isoforms.

We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.

Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P, the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel, ubiquitously expressed PtdIns 4-kinase (PI4Kbeta). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kbeta are characteristic of a type III enzyme. Interestingly, both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi, S., Catt, J. K., and Balla, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5317-5321).

Phosphoinositide 3-kinases (PI(3)Ks) are mainly known for their lipid kinase activity in various cellular functions; however, PI(3)Ks also have protein kinase activity. Non-muscle tropomyosin has been identified as a novel protein target for PI(3)Kγ. Phosphorylation of tropomyosin is required for β-adrenergic receptor internalization, providing new insight into PI(3)K function during clathrin-mediated endocytosis.

VPS34 is a phosphatidylinositol 3-kinase (PI3K) that catalyzes phosphorylation of phosphatidylinositol. In response to nutrients, VPS34 contributes to the activation of mTOR/S6K1 signaling which regulates proliferation, protein synthesis, and cell survival. Intracellular mechanisms leading to VPS34 activation are just beginning to be elucidated. Src is a non-receptor tyrosine kinase that signals downstream of numerous pathways to regulate cell proliferation, survival, and cancer metastasis. Here, we report that Src directly phosphorylates VPS34 leading to activation of VPS34 lipid kinase activity, and that VPS34 lipid kinase activity is required for Src-mediated cellular transformation. Silencing VPS34 specifically inhibits Src-Y527F-induced colony formation in soft agar, but not Ras-G12V-induced colony formation. We have identified two novel VPS34 mutations, which either eliminate lipid kinase activity (KD mutant) or reduce tyrosine phosphorylation (Y231F mutant) by Src-Y527F. When VPS34-KD is stably expressed in Src-Y527F-transformed cells, proliferation and anchorage independent growth are significantly inhibited. Additionally, stable expression of VPS34-KD causes an increased number of binucleate and multinucleate cells, suggesting that VPS34 kinase activity is also required for cytokinesis. Stable expression of VPS34-Y231F in Src-Y527F transformed cells also reduces colony formation. We additionally found that VPS34 protein levels and tyrosine phosphorylation correlate with the tumorigenic activity of breast cancer cell lines. Together these data suggest that Src to VPS34 signaling may contribute to the development and progression of human cancers. Citation Format: Dianne S. Hirsch, Yi Shen, Milos Dokmanovic, Wen Jin Wu. VPS34 lipid kinase activity is positively regulated by Src phosphorylation and is required for Src-mediated cellular transformation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-38. doi:10.1158/1538-7445.AM2013-LB-38

The PI3K/mTOR pathway is involved in promoting tumor cell proliferation, survival and metastasis. The development of new anticancer therapies targeting different components of this pathway has been motivated by the identification of somatic PIK3CA missense mutations as well as the high frequency of loss of negative regulatory proteins such as the tumor suppressor PTEN. In this last case, the abnormal activation of downstream effectors is mediated by PI3Kβ. PTEN deficiency has also been reported to be involved in the resistance to a variety of anticancer therapies. The preceding findings support investigation into selective modulators of the lipid kinase activity of p110β as agents for the treatment of PTEN-deficient tumors, with potentially reduced on-target adverse events described for pan-PI3K inhibitors currently undergoing clinical trials. SAR260301 is a selective PI3Kβ lipid kinase inhibitor that has demonstrated higher potency in vitro on p110β (IC50= 52 nM) than on p110δ and p110α isoforms and no or weak activity against p110γ, mTOR and a wide range of protein kinases (>400 enzymes). The potency and selectivity against the intended target was further confirmed in mechanistic cellular models (IC50 = 32 nM, p110β; 26- and 88-fold selective over p110δ and p110α, respectively). Moreover, SAR260301 also modulates pAkt levels exclusively in PTEN-deficient tumor cell lines with IC50 values ranging from 39 to 310 nM and from 20 to 209 nM for pAkt-S473 and pAkt-T308, respectively. When administered orally, the compound shows dose and time-dependent inhibition of the PI3K pathway in human PTEN-deficient tumor xenografts in mice. In these models, target engagement correlates with compound exposure and is associated with good tolerability. The MAPK pathway also plays an important role in cancer and is often constitutively and simultaneously activated with the PI3K pathway. Furthermore, PTEN loss has been shown to correlate with resistance to MAPK inhibitors. Preclinical data evidencing the ability of SAR260301 to modulate pAkt levels in genetic contexts where PTEN deficiency co-exists with MAPK pathway activation, as well as the effects of combining SAR260301 with MAPK inhibitors will also be presented. The foregoing studies support ongoing Phase I clinical investigation of SAR260301 in patients with PTEN-deficient malignancies. Citation Format: Angela Virone-Oddos, Hélène Bonnevaux, Olivier Lemaitre, Loic Vincent, Franck Halley, Brigitte Demers, Véronique Charrier, Olivier Courtin, Stéphane Guérif, Laurent Besret, Pierre-Yves Abecassis, Sylvaine Cartot-Cotton, Gary Emmons, Alan Roberts, David Compton, Bin Wu, Laurent Schio, Christoph Lengauer, Carlos Garcia-Echeverria. Discovery and characterization of SAR260301, a novel PI3Kβ-selective inhibitor in clinical development for the treatment of PTEN-deficient tumors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3258. doi:10.1158/1538-7445.AM2013-3258

Gain-of-function mutations of the receptor tyrosine kinase c-Kit are frequently seen in several malignanices with the D816V mutation being the most common. In this study we demonstrate that a mutant of c-Kit/D816V that is deficient in binding of the p85 subunit of PI3-kinase (M724A) has lost its ability to induce ligand-independent cell survival and proliferation while c-Kit/D816V induced PI3-kinases activity was only marginally reduced. Furthermore, inhibitors of PI3-kinase were unable to block c-Kit/D816V-induced cell survival. These data suggest that PI3-kinase plays a role in c-Kit/D816V induced cell transformation which is independent of its lipid kinase activity. By mass spectrometry, we identified a heavily tyrosine-phosphorylated 110 kDa protein associated with p85 to be the p110δ subunit of PI3-kinase and the site of phosphorylation as Tyr 523, and the phosphorylation is not dependent on the lipid kinase activity of PI3-kinase. Knockdown of p110δ in c-Kit/D816V expressing Ba/F3 cells led to reduced cell survival and proliferation while the PI3-kinase activity remained almost intact. The c-Kit/D816V expressing Ba/F3 cells lost their ability to form colonies in semi-solid culture if p110δ was knocked down. Additionally, knockdown of p110δ λεδ τo ρεδυχεδ τυμoρ σιζε, τυμoρ ωειγητ ανδ blood vessel formation ωηεν c-Kit/D816V expressing Ba/F3 cells were subcutaneously injected into nude mice. Taken together, these results suggest a kinase independent role of p110δ in the D816Vmutant of c-Kit mediated cell transformation and suggest that p110δ could potentially be a selective therapeutic target for c-Kit/D816V expressing tumors. Citation Format: Jianmin Sun, Sofie Mohlin, Alicia Lundby, Ulf Hellman, Sven Påhlman, Jesper V. Olsen, Lars Rönnstrand. Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2013-4301

PIK3CA, which encodes the catalytic subunit of phosphoinositide 3-kinase PI3Kα (p110α), is frequently mutated in human cancers. PI3Kα consists of a catalytic subunit p110α and one of several regulatory subunits (a major one being p85α). The p110α subunit contains an N-terminal adaptor-binding domain (ABD), a Ras-binding domain (RBD), a C2 domain, a helical domain and a catalytic domain. In the basal state, the regulatory p85 subunit stabilizes the catalytic p110α subunit and inhibits its enzymatic activity. Upon growth factor stimulation, the SH2 domains of p85 bind to the phospho-tyrosine residues on the receptor protein kinases or adaptor proteins such as insulin receptor substrate 1 (IRS1), thereby activating the lipid kinase activity of PI3Kα. Activated PI3Kα converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). The second messenger PIP3 then activates downstream AKT signaling. The majority of these mutations occur at two hot-spots located in the helical (E545K) and the kinase (H1047R) domains. Here, we report that p110α E545K mutant proteins, but not the wild-type and H1047R mutant proteins, directly associate with IRS1 and that this interaction does not require the p85 regulatory subunit of PI3Kα. Disruption of IRS1-p110α E545K specific interaction results in reduction of AKT phosphorylation and slower xenograft tumor growth of a colon cancer cell line harboring this mutation. Moreover, IRS1- p110α E545K interaction stabilizes p110α proteins and brings it cytoplasmic membrane. Our study establishes a new paradigm whereby a mutant protein exerts its oncogenic functions through rewiring an oncogenic signaling pathway. Moreover, we designed an 18 amino acid hydrocarbon-stapled peptide encompassing the p110α E545K mutation that disrupts IRS1-p110α E545K interaction. The stapled mutant peptide reduces AKT phosphorylation in cancer cell lines with a p110α E545K mutation, whereas it has no effect on those cancer cell lines harboring a p110α H1047R mutation. When injected into xenograft tumors, the stapled peptide specifically inhibits growth of tumors with a p110α E545K mutation. Therefore, our data suggest that interference of IRS1-p110α E545K interaction may be exploited for targeted therapy of cancer patients harboring this mutation. The discovery of frequent mutations of PIK3CA in human cancer provides a strong rationale for inhibition of mutated p110α activities for targeted cancer therapy. However, it has remained a challenge to develop p110α isoform-specific inhibitors. Our data suggest that disruption of the interactions between helical domain mutations of p110α and IRS1 may be exploited as a more accessible targeted therapy approach. Citation Format: Yujun Hao, Chao Wang, Weiping Zheng, David Sedwick, Sanford Markowitz, Rob Ewing, Lili Liu, Zhenghe Wang. Gain of direct interaction with IRS1 by the helical domain mutations of p110 alpha is crucial for their oncogenic functions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5155. doi:10.1158/1538-7445.AM2013-5155

Ligand stimulation of the platelet-derived growth factor (PDGF) receptor results in its association with phosphoinositide 3-kinase activity and a corresponding synthesis of 3'-phosphorylated lipids. Early studies that examined this interaction in vivo employed anti-phosphotyrosine antiserum or antiserum against the PDGF receptor. The recent identification of multiple isoforms of both the regulatory and the catalytic subunit of the enzyme have led us to utilize antisera against p85alpha and p110alpha to characterize the association of this particular phosphoinositide 3-kinase complex with the PDGF receptor following ligand stimulation of murine fibroblasts. Both the p85alpha and p110alpha subunits rapidly associated with the ligand-activated receptor resulting in a transient, 2-fold increase in the total pool of p110alpha lipid kinase activity. This association was stable for 15 min after initial stimulation. Subsequently, both subunits began to dissociate from the receptor with similar kinetics. By 60 min this process was complete, demonstrating that p85alpha and p110alpha both associate with the receptor and dissociate from the receptor as a dimeric complex. At this time, marked PDGF receptor down-regulation was observed. Immunoprecipitation from metabolically labeled cells revealed that p85alpha is constitutively phosphorylated on serine residues in quiescent cultures. Upon PDGF stimulation, this phosphorylation upon serine residues was maintained in addition to tyrosine phosphorylation of this subunit. No phosphorylation of the p110alpha subunit was detected in either quiescent or PDGF-stimulated cells. Quantitation of Western blot analysis demonstrated that only 5% of the total pool of p85alpha associated with the PDGF receptor upon ligand stimulation. The 2-fold increase in the lipid kinase activity measured in immunoprecipitates using either anti-p85alpha or anti-p110alpha antiserum therefore reflects a far greater increase in the specific activity of the enzyme upon its association with the PDGF receptor.

To assess the origin of the previously reported higher type II phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity of sickle-red-cell membranes [Rhoda-Hardy-Dessources, M. D., de Neef, R. S., Merault, G. & Giraud, F. (1993) Biochim. Biophys. Acta 1181, 90–96], we have investigated the possible involvement of protein kinase C and tyrosine kinases in the regulation of the lipid kinase activity. Both protein kinase activities were found to be markedly higher in membranes from the pathological cells. When isolated normal-red-cell or sickle-red-cell membranes were assayed, phosphatidylinositol phosphorylation activity was not significantly modified after phorbol ester modulation of protein kinase C. In contrast, stimulation (with sodium orthovanadate) or inhibiton (by tyrphostin) of tyrosine phosphorylation led, respectively, to increased or decreased PtdIns 4-kinase activity in membranes from both cell types. Moreover, immunoprecipitations of membrane extracts from normal and sickle red cells with anti-PtdIns 4-kinase antibody 4C5G, followed by immunoblotting with an anti-phosphotyrosine Ig, revealed a 56-kDa band migrating with PtdIns 4-kinase activity. Taken together, these findings indicate that PtdIns 4-kinase in red blood cells is a phosphotyrosine-containing protein and could be regulated by a mechanism involving tyrosine phosphorylation, and the increase in PtdIns 4-kinase activity of sickle-red-cell membranes is at least in part mediated by their high intrinsic tyrosine kinase activity.

The protein kinase activity of PI3K phosphorylates specific serine residues in growth factor receptors to promote cell survival; these events are constitutively activated in some leukemias., The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer., Author Summary The ability of cells to survive in the absence of proliferation (cell division), differentiation (cell maturation) or activation allows tissues to maintain cell populations that are poised for rapid responses to damage, infections, or other physiological demands. While this “survival-only” response is fundamental to all physiological processes, the underlying mechanisms are not understood. Many growth factors are potent regulators of cell survival through their ability to bind specific cell surface receptors, which in turn activate specialized enzymes called kinases. Phosphoinositide 3-kinase (PI3K) is a dual specificity kinase that is known to be involved in cell survival and malignant transformation, and it is able to phosphorylate both lipid and protein substrates. While the PI3K lipid kinase activity has been extensively studied, the functional significance of its protein kinase activity remains unclear. Here we show that PI3K protein kinase activity can directly phosphorylate growth factor receptors on human hematopoietic (blood) cells to promote a “survival-only” response. We further show that the protein kinase activity of PI3K can be hijacked to result in uncontrolled growth factor receptor phosphorylation and the deregulated survival of leukemic cells. Our studies provide the first evidence that the protein kinase activity of PI3K can control cell survival and that this activity may be deregulated in cancer.

Phosphatidylinositol (PI) 3-kinase is composed of 110 kDa catalytic and 85 kDa regulatory subunits. The 110 kDa subunit has two intrinsic kinase activities, i.e., Mn2+-dependent protein-serine kinase and Mg2+-dependent lipid kinase activities. These intrinsic kinases have been reported to be interdependent: protein-serine kinase phosphorylates the 85 kDa subunit of PI 3-kinase, which upon phosphorylation inhibits the lipid kinase activity of PI 3-kinase. We report here that phosphoinositides can selectively inhibit the protein-serine kinase activity of PI 3-kinase without affecting lipid kinase activity. This inhibition depends on the phosphorylation status of the phosphoinositides, i.e., PI 4,5-bisphosphate > PI 4-phosphate >>PI. Mn2+ (2 mM) protected protein kinase activity from phosphoinositides-mediated inhibition if added prior to interaction of PI 3-kinase with phosphoinositides. On the other hand, Mn2+ (2 mM) inhibited lipid kinase activity independent of its effect on the protein kinase activity of PI 3-kinase. The present study suggests that the protein-serine kinase and the lipid kinase activities of PI 3-kinase can be selectively inhibited by phosphoinositides and Mn2+ respectively.

PI3Ks are lipid kinases that control signaling pathways involved in cell proliferation, motility, cell death and cell invasion. Class I PI3K contains four isoforms, p110α, p110α, p110α and p110α which carry out non redundant signaling functions. The ≤ and ≤ isoforms are ubiquitously expressed, whereas the ≤ and ≤ isoforms are expressed primarily in lymphocytes and engaged in the regulation of immune responses. A gain of function in PI3K signaling is common to many types of human cancer, specifically mutations in PIK3CA which are found in more than 30% of various solid tumor types, including, breast, endometrium, bladder, colorectal cancers as well as lung cancers. As these mutations constitutively activate the lipid kinase activity of the protein, the cancer-specific mutants of p110α appear to be ideal therapeutic targets for anticancer drug development. p110α isoform specific low molecular weight inhibitors could be efficacious against tumors harboring p110alpha mutations and provide an improved safety profile compared to current pan-PI3K modulators. The 2-aminothiazole scaffold proved to be an excellent starting point for the development of potent PI3K inhibitors. Depending on the substitution pattern good PI3Kalpha selectivity can be achieved. Systematic modification of key residues and further optimization of the drug-like and PK properties have led to the identification of NVP-BYL719, a potent and selective PI3Kalpha inhibitor with a promising biological activity. SAR leading to the discovery of NVP-BYL719 and the structural basis for the PI3Kalpha selectivity will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1922. doi:1538-7445.AM2012-1922

In the current study we generated transgenic mice with mammary gland specific- overexpression of PIK3CA wild type and mutant protein with the aim to characterize the effects of activation of PI3K signaling on normal mammary gland and tumor development. PIK3CA encodes p110α, the predominant isoform of the catalytic subunit of class 1A phosphatidylinositol 3-kinase (PI3K), a lipid phosphokinase. The family of PI3Ks provides signaling for diverse cellular functions, including proliferation, metabolism, migration, translation, apoptosis avoidance, and angiogenesis. Disruption of this tightly regulated pathway by gene loss (PTEN), mutation (PIK3CA, AKT1, or less commonly PIK3R1), or amplification (PIK3CA), is one of the most common alterations in human cancers. PIK3CA mutations result in constitutive activation of p110α, increasing lipid kinase activity and resulting in an increase in activated AKT. The majority of mutations in breast cancer occur at three hotspots: E542K and E545K at exon 9, which encode the helical domain, and H1047R at exon 20, which encodes the kinase domain. Previously, it was described that human mammary epithelial cells expressing these alleles grew efficiently in soft agar and as orthotopic tumors in nude mice. In a mouse model in which PI3K is activated by forced recruitment of p110α to the membrane, increased ductal branching, alveolar hyperplasia, intraductal neoplasia and a low frequency of mammary tumors were observed. To date, no in vivo studies have been reported to determine if PIK3CA mutations play a causative role in breast tumorigenesis. By performing whole mount analysis of mammary gland at 8 weeks of age, we have shown that mice harboring E545K mutation showed increased ductal branching, while expression of H1047R (high copy numbers) results in duct enlargement and defects in duct elongation. Two mice harboring low and high copy numbers of H1047R mutation developed mammary tumors at 14 and 16 months of age respectively. Histological analysis revealed that both were carcinomas with tubular, papillary and comedo features. In conclusion, transgenic mice with mammary gland specific- overexpression of PIK3CA wild type and hot spot mutations were generated. In transgenic mouse studies, we show that PI3K signaling activation appears to alter normal mammary gland development and initiate mammary tumor formation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3255.

Background: Failure to induce apoptosis is a major limitation of conventional chemotherapy for lung cancer. Resistance to apoptosis contributes to this chemoresistant phenotype. New therapeutic strategies are therefore required to bypass block in apoptosis. Obatoclax (obx) is a small molecule inhibitor of pro-survival BCL-2 family proteins currently being clinically evaluated in both small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). However, little is known regarding the molecular mechanisms that might underlie sensitivity and resistance to obx. We are addressing this important question by exploring the pharmacodynamics of obx in NSCLC. Methods: To isolate mitochondria harvested cells were homogenized in ice-cold isolation buffer and the resulting homogenate subjected to differential centrifugation to isolate the mitochondrial fraction. To conduct shRNA transfections, shRNA plasmids targeted against BAX, BAK, BIM and beclin1 were purchased from SA Biosciences (Frederick, MD, USA). H460 cells were transfected with each plasmid and cells were subjected to at least two rounds of selection to create stable clones. For transmission electron microscopy, cells were fixed in double strength TEM fixative (4% paraformaldehyde, 2.5% glutaraldehyde) in 0.1M sodium cacodylate buffer. Samples were analyzed using standard EM procedures. Specific hVps34 Activity was measured in cells deprived of amino acids for 2 hrs in EBSS. Cells were lysed and subjected to immunoprecipitation with anti-hVps34 antibody, and immunocomplexes were assayed for hVps34 lipid kinase activity. Results: shRNA downregulation of BAX/BAK (H460shBAX/BAK) inhibited obx induced caspase 9/3 cleavage, PARP activation, and mitochondrial outer membrane permeabilization. However obx was equally effective in reducing clonogenic survival, and short term viability (48–72 hrs). Obx induced prolific cytoplasmic vacuolation evident by TEM, and processing of LC3I to LC3II was evident in NSCLC H460 and H1975 cells, and SCLC H196 and H146 cells suggesting induction of autophagy. H460shBAX/BAK clones also displayed unaltered LC3 processing. To determine whether LC3 processing following obx was regulated by beclin1, stable knockdown was acomplished using shRNA. 3 clones were selected and exhibited reduced hVpS34 activity, however obatoclax induced equal clonogenic survival and LC3 processing compared to NT shRNA controls. Neither LC3 nor PARP processing was inhibited by 3MA or wortmannin. Also we observed loss of beclin1 expression following obx. Activation of JNK and ERK have been implicated in beclin1 indpendent mitophagy, while JNK is also a known activator of BAX and BIM. Obx induced phosphorylation of JNK but not ERK, and inhibition of pJNK blocked PARP cleavage. Also, stable knockdown of BIM by shRNA did not significantly alter obx EC50 values. In H727 NSCLC cells resistant to obx induced apoptosis, JNK phosphorylation and LC3II formation was observed but to a significantly lesser extent than sensitive cells. Summary: JNK phosphorylation regulates obatoclax induced apoptosis, and this activity is reduced in NSCLC cells resistant to obx. LC3 processing was independent of beclin1, thus the roles of Atg5 and 7 in regulating obx induced LC3 processing and cell fate are ongoing and will be presented. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B30.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical component of intracellular signaling cascades as well as actin dynamics. It is synthesized by the phosphorylation of PI4P by one of three isoforms of PIP5KI (α,β, or γ). We found that the targeted disruption of PIP5KIγ results in early prenatal mortality that prevented studies of bone marrow or liver hematopoietic cells. However, we were able to analyze yolk sac progenitor cells differentiated ex vivo into megakaryocytes. Imaging in the electron microscope showed that PIP5KIγ-null megakaryocytes have normal architectural appearance of their microtubules, and formed proplatelets and platelets. This demonstrates that PIP5KIγ is not essential for the development of megakaryocytes, proplatelets, or platelets. PIP5KIγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we found that wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon a fibrinogen matrix. In contrast, PIP5KIγ-null megakaryocytes continuously and slowly extended and retracted membrane blebs, rather than lamellipodia. We analyzed whether PIP5KIγ-null megakaryocytes have a defect in their ability to anchor their membranes to the cytoskeleton accounting for the blebbing phenotype by using laser tweezers to pull the cell membrane apart from the cytoskeleton. Fibrinogen-coated beads bound both wild type and PIP5KIγ-null megakaryocytes. Yet wild type cells had rigid membranes that resisted stretching by trapped beads that were pulled by laser tweezers. In contrast, the majority of PIP5KIγ-null megakaryocytes had flexible membranes that were easily stretched by pulling on the fibrinogen-coated beads, and ultimately allowed membrane tethers to form. We found that the membrane phenotype was completely reverted by adding back wild type PIP5KIγ, but not by a catalytically inactive PIP5KIγ mutant. This shows that the lipid kinase activity of PIP5KIγ regulates the membrane phenotype. Surprisingly, PIP5KIβ-null megakaryocytes have at least as great of a defect in PIP2 synthesis but lack this membrane abnormality. Furthermore, overexpression of PIP5KIβ in PIP5KIγ-null megakaryocytes fails to rescue the cytoskeletal defect. Adding back a splice variant of PIP5KIγ that lacks the binding site for talin, also fails to revert this cytoskeletal defect present in PIP5KIγ-null cells. This suggests that talin binding and PIP2 synthesized specifically by the PIP5KIγ isoform are required for the cytoskeletal organization of megakaryocytes. Given the proposed role of PIP2 on the regulation of the demarcation membrane system (DMS), we expressed GFP-fused to β3-integrin and followed its trafficking from the DMS to the surface membrane. GFP-β3 situated on the DMS externalized to the cellular membrane within minutes of plating a wild type megakaryocyte upon immobilized fibrinogen. In megakaryocytes lacking PIP5KIγ, GFP-β3 situated on the DMS poorly externalized, and was barely detectable on the cell membrane 1 hour after adhering to immobilized fibrinogen. This shows that trafficking of αIIbβ3 from the DMS to the cell membrane requires PIP5KIγ. Together, this demonstrates that spatially restricted production of PIP2 by PIP5KIγ is required to preserve the integrity of the membrane cytoskeleton, and that PIP2 synthesized by PIP5KIβ in megakaryocytes can not contribute to this process.

Excess levels of circulating amino acids (AAs) play a causal role in specific human pathologies, including obesity and type 2 diabetes. Moreover, obesity and diabetes are contributing factors in the development of cancer, with recent studies suggesting that this link is mediated in part by AA activation of mammalian target of rapamycin (mTOR) Complex 1. AAs appear to mediate this response through class III phosphatidylinositol 3-kinase (PI3K), or human vacuolar protein sorting 34 (hVps34), rather than through the canonical class I PI3K pathway used by growth factors and hormones. Here we show that AAs induce a rise in intracellular Ca(2+) ([Ca(2+)](i)), which triggers mTOR Complex 1 and hVps34 activation. We demonstrate that the rise in [Ca(2+)](i) increases the direct binding of Ca(2+)/calmodulin (CaM) to an evolutionarily conserved motif in hVps34 that is required for lipid kinase activity and increased mTOR Complex 1 signaling. These findings have important implications regarding the basic signaling mechanisms linking metabolic disorders with cancer progression.

The p110α catalytic subunit ( PIK3CA ) is one of the most frequently mutated genes in cancer. We have examined the activation of the wild-type p110α/p85α and a spectrum of oncogenic mutants using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We find that for the wild-type enzyme, the natural transition from an inactive cytosolic conformation to an activated form on membranes entails four distinct events. Analysis of oncogenic mutations shows that all up-regulate the enzyme by enhancing one or more of these dynamic events. We provide the first insight into the activation mechanism by mutations in the linker between the adapter-binding domain (ABD) and the Ras-binding domain (RBD) (G106V and G118D). These mutations, which are common in endometrial cancers, enhance two of the natural activation events: movement of the ABD and ABD–RBD linker relative to the rest of the catalytic subunit and breaking the C2–iSH2 interface on binding membranes. C2 domain mutants (N345K and C420R) also mimic these events, even in the absence of membranes. A third event is breaking the nSH2–helical domain contact caused by phosphotyrosine-containing peptides binding to the enzyme, which is mimicked by a helical domain mutation (E545K). Interaction of the C lobe of the kinase domain with membranes is the fourth activation event, and is potentiated by kinase domain mutations (e.g., H1047R). All mutations increased lipid binding and basal activity, even mutants distant from the membrane surface. Our results elucidate a unifying mechanism in which diverse PIK3CA mutations stimulate lipid kinase activity by facilitating allosteric motions required for catalysis on membranes.

Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

Vanadate increases the initial phosphatidylinositolphosphate formation rate as well as the steady state level of the above lipid phosphate when phosphatidylinositol associated with the isolated Ca2+ transport ATPase is phosphorylated either by the membrane bound endogeneously present phosphatidylinositol-kinase or by exogeneously added lipid kinase. Employing an ultrasonicated mixture of pure phosphatidylinositol and Triton X 100 (without membrane proteins) no vanadate effect can be seen. This vanadate effect is probably not mediated through the lipid kinase activity, but more likely, through conformational changes of the Ca2+ transport ATPase protein. Such conformational changes would lead to a higher degree of phosphatidylinositol exposed on the Ca2+ transport ATPase and thus a higher substrate concentration. Consequently, the initial phosphatidylinositolphosphate formation rate and steady state level increase.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidate (PA) and choline. PLD activity in mammalian cells is low and is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors. Two mammalian PLD enzymes (PLD1 and PLD2) have been cloned and their intracellular regulators identified as ARF and Rho proteins, protein kinase Calpha as well as the lipid, phosphatidylinositol bisphosphate (PIP2). I discuss the regulation of these enzymes by cell surface receptors, their cellular localisation and the potential function of PA as a second messenger. Evidence is presented for a role of PA in regulating the lipid kinase activity of PIP 5-kinase, an enzyme that synthesises PIP2. A signalling role of phospholipase D via PA and indirectly via PIP2 in regulating membrane traffic and actin dynamics is indicated by the available data.

Aims/hypothesis. Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. Methods. Phosphoinositide 3-kinase p85a regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85a were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85a-GST fusion proteins were examined by surface plasmon resonance. Results. The common p85a variant, Met 326 I1e, was identified in 9 of the 20 subjects. Functional studies of the Met 326 Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85a. A novel heterozygous mutation, Arg 409 Gln, was detected in one subject. Within the proband’s family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg 409 Gln p85a subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15 %, p < 0.05, n = 5). The recruitment of Arg 409 Gln p85a into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85a showed identical binding to phosphopeptides in surface plasmon resonance studies. Conclusion/interpretation. Mutations in p85a are uncommon in subjects with syndromes of severe insulin resistance. The Met 326 Ile p85a variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg 409 Gln mutant suggests that it could contribute to the insulin resistance seen in this family. [Diabetologia (2000) 43: 321‐331]

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.

Mammals have genes coding for three PI5P4Ks (PtdIns5P 4-kinases), and these have different cellular localizations, tissue distributions and lipid kinase activities. We describe in the present paper a detailed molecular exploration of human PI5P4Ks α, β and γ, as well as their fly and worm homologues, to understand how and why these differences came to be. The intrinsic ATPase activities of the three isoforms are very similar, and we show that differences in their G-loop regions can account for much of their wide differences in lipid kinase activity. We have also undertaken an extensive in silico evolutionary study of the PI5P4K family, and show experimentally that the single PI5P4K homologues from Caenorhabditis elegans and Drosophila melanogaster are as widely different in activity as the most divergent mammalian isoforms. Finally we show that the close association of PI5P4Ks α and γ is a true heterodimerization, and not a higher oligomer association of homodimers. We reveal that structural modelling is consistent with this and with the apparently random heterodimerization that we had earlier observed between PI5P4Kα and PI5P4Kβ [Wang, Bond, Letcher, Richardson, Lilley, Irvine and Clarke (2010), Biochem. J. 430, 215–221]. Overall the molecular diversity of mammalian PI5P4Ks explains much of their properties and behaviour, but their physiological functionality remains elusive.