Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival

Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85alpha gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85alpha, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85alpha represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.     

Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling

Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85 alpha and p85 beta, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1-/- (p85 alpha-/-) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30% decrease in Akt activity, leading to decreased insulin-induced glucose uptake and anti-apoptosis. Pik3r2-/- (p85 beta-/-) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85 beta-/- cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85 alpha-/- cells and p85 beta-/- cells exhibit significantly increased insulin-induced glycogen synthase activation. p85 alpha-/- cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85 alpha, p85 beta, or a p85 mutant that does not bind to p110, indicating the existence of p85-dependent, but PI 3-kinase-independent, signaling pathway. Furthermore, a reduction of p85 beta specifically increases insulin receptor substrate-2 phosphorylation. Thus, p85 alpha and p85 beta modulate PI 3-kinase-dependent signaling by multiple mechanisms and transmit signals independent of PI 3-kinase activation.     

Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha

The p85alpha regulatory subunit of class I(A) phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50alpha and p55alpha. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85alpha. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85alpha-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85beta subunit. Furthermore, p85alpha-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G(0)/G(1) cell cycle arrest and up-regulation of p27(KIP), whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85alpha via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85beta signaling was lost in p85alpha-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85alpha in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.     

The mammalian class 3 PI3K (PIK3C3) is required for early embryogenesis and cell proliferation

The Pik3c3 gene encodes an 887 amino acid lipid kinase, phosphoinositide-3-kinase class 3 (PIK3C3). PIK3C3 is known to regulate various intracellular membrane trafficking events. However, little is known about its functions during early embryogenesis in mammals. To investigate the function of PIK3C3 in vivo, we generated Pik3c3 null mice. We show here that Pik3c3 heterozygous are normal and fertile. In contrast, Pik3c3 homozygous mutants are embryonic lethal and die between E7.5 and E8.5 of embryogenesis. Mutant embryos are poorly developed with no evidence of mesoderm formation, and suffer from severely reduced cell proliferations. Cell proliferation defect is also evident in vitro, where mutant blastocysts in culture fail to give rise to typical colonies formed by inner cell mass. Electron microscopic analysis revealed that epiblast cells in mutant embryos appear normal, whereas the visceral endoderm cells contain larger vesicles inside the lipid droplets. Finally, we provide evidence that mTOR signaling is drastically reduced in Pik3c3 null embryos, which could be a major contributor to the observed proliferation and embryogenesis defects.     

Membrane localization of all class I PI 3-kinase isoforms suppresses c-Myc-induced apoptosis in Rat1 fibroblasts via Akt

Phosphoinositide 3'-kinases (PI3Ks) constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. PI3Ks are heterodimers made up of four different 110-kDa catalytic subunits (p110alpha, p110beta, p110gamma, and p110delta) and a smaller regulatory subunit. Despite a clear implication of PI3Ks in survival signaling, the contribution of the individual PI3K isoforms has not been elucidated. To address this issue, we generated Rat1 fibroblasts that co-express c-Myc and membrane targeted derivates of the different p110 isoforms. Here we present data for the first time showing that activation of PI3-kinase signaling through membrane localization of p110beta, p110gamma, and p110delta protects c-Myc overexpressing Rat1 fibroblasts from apoptosis caused by serum deprivation like it has been described for p110alpha. Expression of each p110 isoform reduces significantly caspase-3 like activity in this apoptosis model. Decreased caspase-3 activity correlates with the increase in Akt phosphorylation in cells that contain one of the myristoylated p110 isoforms. p110 isoform-mediated protection from cell death was abrogated upon expression of a kinase-negative version of Akt.     

Pik3ip1 Modulates Cardiac Hypertrophy by Inhibiting PI3K Pathway

Cardiac hypertrophy is an adaptive response to various physiological and pathological stimuli. Phosphoinositide-3 kinase (PI3K) is a highly conserved lipid kinase involved in physiological cardiac hypertrophy (PHH). PI3K interacting protein1 (Pik3ip1) shares homology with the p85 regulatory subunit of PI3K and is known to interact with the p110 catalytic subunit of PI3K, leading to attenuation of PI3K activity in liver and immune cells. However, the role of Pik3ip1 in the heart remains unknown. In the present study, the effects of Pik3ip1 on cardiac hypertrophy were examined. We found that the expression level of Pik3ip1 was markedly higher in cardiomyocytes than in fibroblasts. The interaction of Pik3ip1 with the p110a subunit of PI3K in the heart was identified by immunoprecipitation using neonatal rat cardiomyocytes (NRCM). Approximately 35% knockdown of Pik3ip1 was sufficient to induce myocardial hypertrophy. Pik3ip1 deficiency was shown to lead to activation of PI3K/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling pathway, increasing protein synthesis and cell size. However, adenovirus-mediated overexpression of Pik3ip1 attenuated PI3K-mediated cardiac hypertrophy. Pik3ip1 was upregulated by PHH due to swimming training, but not by pathological cardiac hypertrophy (PAH) due to pressure-overload, suggesting that Pik3ip1 plays a compensatory negative role for PHH. Collectively, our results elucidate the mechanisms for the roles of Pik3ip1 in PI3K/AKT signaling pathway.     

Structure and function of phosphatidylinositol-3,4 kinase

Activation of phosphatidylinositol (PI)-kinase is involved in the regulation of a wide array of cellular activities. The enzyme exists as a dimer, consisting of a catalytic and a regulatory subunit. Five isoforms of the regulatory subunit have been identified and classified into three groups comprising respectively 85-kDa, 55-kDa, and 50-kDa proteins. Structural differences in the N-terminal regions of the different group members contribute to defining their binding specificity, their subcellular distributions, and their capacity to activate the 110-kDa catalytic subunit. Two widely distributed isoforms of the catalytic subunit have been identified-p110alpha and p110beta. Despite the fact that they bind to the p85alpha regulatory subunit similarly, p110alpha and p110beta appear to have separate functions within cells and to be activated by different stimuli. Moreover, although p85/p110 PI-kinase almost exclusively phosphorylates the D-3 position of the inositol ring in phosphoinositides when purified PI is used as a substrate in vitro, it appears to phosphorylate the D-4 position with similar or higher efficiency in vivo. Thus, it is highly probable that p85/p110 PI-kinase transmits signals to downstream targets via both D-3- and D-4-phosphorylated phosphoinositides.     

A Role for phosphoinositide 3-kinase in the control of cell division and survival during retinal development

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, as p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. Here, we examine the role of PI3K in mouse retina development by expressing an activating form of PI3K regulatory subunit, p65(PI3K), as a transgene in the retina. Mice expressing p65(PI3K) showed severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolved into infoldings of adult retinal cell layers. These changes correlated with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina was that of photoreceptors, which correlated with selectively increased survival of these cells at developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo.     

The class IA phosphatidylinositol 3-kinase p110-beta subunit is a positive regulator of autophagy

Autophagy is an evolutionarily conserved cell renewal process that depends on phosphatidylinositol 3-phosphate (PtdIns(3)P). In metazoans, autophagy is inhibited by PtdIns(3,4,5)P(3), the product of class IA PI3Ks, which mediates the activation of the Akt-TOR kinase cascade. However, the precise function of class IA PI3Ks in autophagy remains undetermined. Class IA PI3Ks are heterodimeric proteins consisting of an 85-kD regulatory subunit and a 110-kD catalytic subunit. Here we show that the class IA p110-β catalytic subunit is a positive regulator of autophagy. Genetic deletion of p110-β results in impaired autophagy in mouse embryonic fibroblasts, liver, and heart. p110-β does not promote autophagy by affecting the Akt-TOR pathway. Rather, it associates with the autophagy-promoting Vps34-Vps15-Beclin 1-Atg14L complex and facilitates the generation of cellular PtdIns(3)P. Our results unveil a previously unknown function for p110-β as a positive regulator of autophagy in multicellular organisms.     

A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo.

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.     

Class I phosphoinositide 3-kinase p110beta is required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by macrophages

Phosphoinositide 3-kinases (PI3Ks) play an important role in a variety of cellular functions, including phagocytosis. PI3Ks are activated during phagocytosis induced by several receptors and have been shown to be required for phagocytosis through the use of inhibitors such as wortmannin and LY294002. Mammalian cells have multiple isoforms of PI3K, and the role of the individual isoforms during phagocytosis has not been addressed. The class I PI3Ks consist of a catalytic p110 isoform associated with a regulatory subunit. Mammals have three genes for the class IA p110 subunits encoding p110alpha, p110beta, and p110delta and one gene for the class IB p110 subunit encoding p110gamma. Here we report a specific recruitment of p110beta and p110delta (but not p110alpha) isoforms to the nascent phagosome during apoptotic cell phagocytosis by fibroblasts. By microinjecting inhibitory antibodies specific to class IA p110 subunits, we have shown that p110beta is the major isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary mouse macrophages. Macrophages from mice expressing a catalytically inactive form of p110delta showed no defect in the phagocytosis of apoptotic cells and IgG-opsonized particles, confirming the lack of a major role for p110delta in this process. Similarly, p110gamma-deficient macrophages phagocytosed apoptotic cells normally. Our findings demonstrate that p110beta is the major class I catalytic isoform required for apoptotic cell and Fcgamma receptor-mediated phagocytosis by primary macrophages.     

Identification and characterization of a novel high-molecular-weight form of the integrin alpha 3 subunit.

The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit.     

Conditional disruption of ubiquitous calpains in the mouse

Ubiquitous mu- and m-calpain proteases are implicated in development and apoptosis. They are heterodimers consisting of 80-kDa catalytic subunits encoded by capn1 and capn2, respectively, and a common 28-kDa regulatory subunit encoded by capn4. The regulatory subunit is required to maintain stability and activity of mu- and m-calpains; thus, genetic disruption of capn4 was predicted to eliminate both calpain activities. Germline disruption of capn4 caused embryonic lethality, hampering the use of those mouse models to explore physiological calpain functions. Here we describe a loxP/cre conditional capn4 targeted mouse model that enables tissue-specific and temporal deletion of calpain activity. Disruption of the floxed capn4 gene using a ubiquitous cytomegalovirus promoter driven Cre recombinase transgene led to midgestation embryonic lethality. Fibroblasts from these embryos lacked detectable regulatory subunit expression, had reduced levels of the mu- and m-calpain catalytic subunits, and had no detectable mu- and m-calpain activities. These defects were corrected with a capn4-encoding lentivirus.     

Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B.

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.     

Class IA phosphoinositide 3-kinase modulates basal lymphocyte motility in the lymph node

Recruitment of PI3K to the cell membrane is an indispensable step in normal lymphocyte proliferation and activation. In this study we identify PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the class IA PI3K regulatory subunits p85alpha and p85beta also exhibited reduced velocities, with the magnitude of reduction depending upon both cell type and isoform specificity. B cells deficient in p85alpha exhibited gross morphological abnormalities that were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in regulating basal lymphocyte motility and that p85alpha regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing, and homeostatic localization of mature resting B cells.     

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors.

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110 alpha or p110 beta). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to alpha beta gamma-trimeric G proteins also stimulate the lipid kinase activity of a novel p110 gamma isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110 gamma PI 3-kinase appears to be mediated through the beta gamma subunits of the G protein (G beta gamma). In addition, rat liver heterodimeric PI 3-kinases containing the p110 beta catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus G beta gamma. Such enzymatic properties were also observed with a recombinant p110 beta/p85 alpha expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110 alpha and p85 in the same rat liver, together with a recombinant p110 alpha/p85 alpha, was not activated by G beta gamma, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide (N-formyl-Met-Leu-Phe) plus insulin (or Fc gamma II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110 beta catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a 'cross-talk' enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.     

Human Tumor Mutants in PI3K p110α Subunit

The PI3K-Akt pathway is frequently upregulated in human tumors. Recently, somatic mutations of PIK3CA, encoding p110α catalytic subunit of Class IA PI3Ks, have been found in various cancers. The two most common types of p110α mutants, those in the helical and kinase domains, have been shown to be very potent in Akt activation and oncogenic transformation by several groups. Notably these common mutations may not enhance recruitment of p110α to the plasma membrane where its substrates are located. We have investigated the effect of membrane localization on common PIK3CA tumor mutants via myristoylation. In addition we have studied a third class of less frequent mutants in the p85-binding domain, in an attempt to gain insight into p85’s inhibitory effect on p110α. This article briefly reviews and extends the literature on mutant forms of p110α.     

The p85alpha regulatory subunit of class IA phosphoinositide 3-kinase regulates beta-selection in thymocyte development

We examined the role of class IA PI3K in pre-TCR controlled beta-selection and TCR-controlled positive/negative selection in thymic development. Using mice deficient for p85alpha, a major regulatory subunit of the class IA PI3K family, the role of class IA PI3K in beta-selection was examined by injection of anti-CD3epsilon mAb into p85alpha(-/-)Rag-2(-/-) mice, which mimics pre-TCR signals. Transition of CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP) thymocytes triggered by anti-CD3epsilon mAb was significantly impaired in p85alpha(-/-)Rag-2(-/-) compared with p85alpha(+/-)Rag-2(-/-) mice. Furthermore, DP cell numbers were lower in p85alpha(-/-)DO11.10/Rag-2(-/-) TCR-transgenic mice than in DO11.10/Rag-2(-/-) mice. In addition, inhibition by IC87114 of the major class IA PI3K catalytic subunit expressed in lymphocytes, p110delta, blocked transition of DN to DP cells in embryonic day 14.5 fetal thymic organ culture without affecting cell viability. In the absence of phosphatase and tensin homolog deleted on chromosome 10, where class IA PI3K signals would be amplified, the DN to DP transition was accelerated. In contrast, neither positive nor negative selection in Rag-2(-/-)TCR-transgenic mice was perturbed by the lack of p85alpha. These findings establish an important function of class IA PI3K in the pre-TCR-controlled developmental transition of DN to DP thymocytes.     

Structural and mechanistic insights provided by single particle cryo-EM analysis of phosphoinositide 3-kinase (PI3Kα)

Recent cryo-electron microscopic (cryo-EM) investigations have succeeded in the analysis of various structural conformations and functional states of PI3Kα, a dimer consisting of the catalytic subunit p110α and the regulatory subunit p85α of class IA of phosphoinositide 3-kinase. High resolution structures have been obtained of the unliganded and of BYL-719-bound PI3Kα. The latter provides information on excessively flexible domains of p85α that are then further analyzed with nanobodies and CXMS (chemical cross-linking, digestion and mass spectrometry). Analysis of p110α helical and kinase domain mutations reveals mutant-specific features that can be linked to the gain of function in enzymatic and signaling activities.     

An adenosine triphosphatase of the sucrose nonfermenting 2 family, androgen receptor-interacting protein 4, is essential for mouse embryonic development and cell proliferation

An adenosine triphosphatase of the sucrose nonfermenting 2 protein family, androgen receptor-interacting protein 4 (ARIP4), modulates androgen receptor activity. To elucidate receptor-dependent and -independent functions of ARIP4, we have analyzed Arip4 gene-targeted mice. Heterozygous Arip4 mutants were normal. Arip4 is expressed mainly in the neural tube and limb buds during early embryonic development. Arip4-/- embryos were abnormal already at embryonic d 9.5 (E9.5) and died by E11.5. At E9.5 and E10.5, almost all major tissues of Arip4-null embryos were proportionally smaller than those of wild-type embryos, and the neural tube was shrunk in some Arip4-/- embryos. Dramatically reduced cell proliferation and increased apoptosis were observed in E9.5 and E10.5 Arip4-null embryos. Mouse embryonic fibroblasts (MEFs) isolated from Arip4-/- embryos ceased to grow after two to three passages and exhibited increased apoptosis and decreased DNA synthesis compared with wild-type MEFs. Comparison of gene expression profiles of Arip4-/- and wild-type MEFs at E9.5 revealed that putative ARIP4 target genes are involved in cell growth and proliferation, apoptosis, cell death, DNA replication and repair, and development. Collectively, ARIP4 plays an essential role in mouse embryonic development and cell proliferation, and it appears to coordinate multiple essential biological processes, possibly through a complex chromatin remodeling system.