Phosphotidylinositol 3 kinase 92E
Although nitric oxide (NO) has potent antiplatelet actions, the signaling pathways affected by NO in the platelet are poorly understood.
Since NO can induce platelet disaggregation and phosphoinositide 3-kinase (PI3-kinase) activation renders aggregation irreversible, a
test was made of the hypothesis that NO exerts its antiplatelet effects at least in part by inhibiting PI3-kinase. The results demonstrate that the NO
donor S-nitrosoglutathione (S-NO-glutathione) inhibits the stimulation of PI3-kinase associated with tyrosine-phosphorylated proteins
and of p85/PI3-kinase associated with the SRC family kinase member LYN following the exposure of platelets to thrombin
receptor-activating peptide. The activation of LYN-associated PI3-kinase is unrelated to changes in the amount of PI3-kinase
physically associated with LYN signaling complexes but requires the activation of LYN and other tyrosine kinases. The cyclic
GMP-dependent kinase activator 8-bromo-cyclic GMP has similar effects on PI3-kinase activity, consistent with a model in which the
cyclic nucleotide mediates the effects of NO. Additional studies have shown that wortmannin and S-NO-glutathione have additive
inhibitory effects on thrombin receptor-activating peptide-induced platelet aggregation and the surface expression of platelet activation
markers. These data provide evidence of a distinct and novel mechanism for the inhibitory effects of NO on platelet function (Pigazzi, 1999).
Dictyostelium SHK1 is a novel dual-specificity kinase that contains an SH2 domain in its C-terminal region. SHK1 is required for proper chemotaxis and phagocytosis. Mutant shk1 null cells lack polarity, move very slowly, and exhibit an elevated and temporally extended chemoattractant-mediated activation of the kinase Akt/PKB. GFP fusions of the PH domain of Akt/PKB or the PH-domain-containing protein CRAC, which become transiently associated with the plasma membrane after a global stimulation with a chemoattractant, remain associated with the plasma membrane for an extended period of time in shk1 null cells. These results suggest
that SHK1 is a negative regulator of the PI3K (phosphatidylinositol-3 kinase) pathway. Furthermore, when a chemoattractant gradient is applied to a wild-type cell,
these PH-domain-containing proteins and the F-actin-binding protein coronin localize to its leading edge, but in an shk1 null cell they become randomly associated
with the plasma membrane and cortex, irrespective of the direction of the chemoattractant gradient, suggesting that SHK1 is required for the proper spatiotemporal control of F-actin levels in chemotaxing cells. Consistent with such functions, SHK1 is localized at the plasma membrane/cortex, and its SH2 domain is
required for this localization and the proper function of SHK1 (Moniakis, 2001).
No SHK1 homolog has been in the databases of other organisms. However, this is not unexpected, because Dictyostelium protein kinases capable of phosphorylating on tyrosine residues, even those that are monospecific tyrosine kinases, tend not to be closely related to metazoan kinases. The SHK1 SH2 domain is most closely
related to that of the p110 PI3K adaptor p55. This may be a chance occurrence, but it may also indicate an evolutionary relationship between the mechanisms by which PI3K pathways are regulated in Dictyostelium and mammalian cells. The PI3K-dependent activation of PKB by chemoattractants in Dictyostelium is probably most closely related to activation of PKB by chemokines through the G-protein-linked PI3K, PI3Kgamma, in leukocytes. Discovering whether SHK1 has more direct parallels to proteins in mammalian cells will require a more detailed characterization of the PI3K pathways (Moniakis, 2001).
A major challenge in cancer genetics is to determine which low-frequency somatic mutations are drivers of tumorigenesis. This study interrogated the genomes of 7,651 diverse human cancers, and inactivating mutations were found in the homeodomain transcription factor gene CUX1 (cut-like homeobox 1) in ~1-5% of various tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, CUX1 was validated as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. It was demonstrated that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth and susceptibility to PI3K-AKT inhibition. Thus, these complementary approaches identify CUX1CUX1 as a pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors (Wong 2014).
Activation of phosphatidylinositide 3'-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the
inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through
phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 [p90(Rsk)], p70(S6kinase),
and also PKB have all been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation, the role of PKB has been investigated by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at
expression levels similar to wild-type PKB), because currently no such mutant has been reported. The PKB mutant (PKB-CAAX) acts as an efficient
inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct
interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB,
gagPKB, induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. These results demonstrate that PKB activation is both necessary and
sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through
direct interaction, since both proteins co-immunoprecipitate. This interaction also results in a translocation of GSK-3 to the membrane in cells expressing transforming
gagPKB (van Weeren 1998).
Three members have been identified in the protein kinase B (PKB) family (see Drosophila Akt1): Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have
demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of
AKT2 is still not well understood. The activation of AKT2 has been demonstrated with several growth factors, including epidermal growth factor, insulin-like growth
factor 1, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin, in human ovarian epithelial cancer cells. The kinase
activity and the phosphorylation of AKT2 are induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and
dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activate AKT2, and
this activation is not significantly enhanced by growth factor stimulation but is abrogated by wortmannin. These results indicate that AKT2 is a downstream
target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further
evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies (Liu, 1998).
Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1: see Drosophila RPS6-p70-protein kinase) activation is mediated by protein
kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively
membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. The importance of
PKB signaling in S6K1 activation was examined. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation
initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and
-sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants,
whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active,
constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted
allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation
and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of
constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced
4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane
localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independent
of its intracellular location (Dufner, 1999).
Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal
roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the
mechanisms underlying their regulation. The activation of phosphatidylinositol 3 (PI3) kinase by extracellular
growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass
of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction,
phosphorylates recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and
FKHR1(T24A/S253A) are resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These
results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus (Biggs, 1999).
Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where
ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is
phosphatidylinositol 3-kinase). However, these two signaling pathways exert opposing effects on muscle
cell hypertrophy. Manipulation of these pathways during muscle differentiation indicates that inhibition of the
Ras-Raf-MEK-ERK pathway promotes differentiation, whereas inhibition of PI3K blocks differentiation. However, the roles of these two pathways in the
process of skeletal muscle hypertrophy has not previously been evaluated. C2C12 myoblasts normally proliferate and are mononucleated. When deprived of serum at confluence, they fuse and differentiate into postmitotic,
elongated, and multinucleated myotubes. The hypertrophic action of insulin-like growth factor-1 (IGF-1) on muscle cells in vivo is mimicked by the addition
of IGF-1 during the differentiation of C2C12 myotubes in vitro, resulting in the generation of thicker myotubes. In addition to inducing hypertrophy of
myotubes in vivo, IGF-1 has been shown to activate both the Raf-MEK-ERK pathway and the PI3K-Akt pathway. The roles of these two pathways in the differentiation and hypertrophy of C2C12 myotubes were examined by genetic manipulation.
Expression of a constitutively active form of Raf (c.a.-Raf) results in the generation of smaller and thinner myotubes, whereas expression of a dominant
negative form of Raf (d.n.-Raf) results in markedly thicker myotubes. Thus, inhibition of the Raf-MEK-ERK pathway induced a hypertrophic
phenotype similar to that elicited by IGF-1 treatment. In contrast, activation of the Akt pathway by expression of a constitutively active form of Akt
(c.a.-Akt) results in a hypertrophic phenotype more pronounced than that observed with d.n.-Raf and characterized by multinucleated myotubes that are
both thickened and shortened. Thus, genetic manipulation of the Raf-MEK-ERK and PI3K-Akt pathways reveals opposing phenotypic effects of these
pathways during muscle differentiation, with the Raf-MEK-ERK pathway inhibiting development of the hypertrophic phenotype and the PI3K-Akt pathway
promoting it. The PI3K-Akt pathway inhibits the Raf-MEK-ERK pathway; this
cross-regulation depends on the differentiation state of the cell: Akt activation inhibits the Raf-MEK-ERK pathway in
differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlates with
its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex (Rommel, 1999).
The signaling pathway comprising Raf, MEK (mitogen-activated protein kinase, or ERK kinase), and ERK (extracellular signal-regulated kinase) lies downstream of
the small guanine nucleotide binding protein Ras and mediates several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest,
differentiation, and senescence, depending on the duration and strength of the external stimulus and on cell type. Another pathway that lies downstream of Ras
includes phosphatidylinositol (PI) 3-kinase and Akt (or protein kinase B) and also regulates these cellular responses, acting either synergistically with or in
opposition to the Raf pathway. Coordination of the two pathways in a single cellular response may depend on cell type or the stage of differentiation. Akt interacts with Raf and phosphorylates this protein at a highly conserved serine residue in its regulatory
domain in vivo. This phosphorylation of Raf by Akt inhibits activation of the Raf-MEK-ERK signaling pathway and
shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations
provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt. These results demonstrate that Akt antagonizes Raf activity by direct phosphorylation of Ser259. This modification creates a binding site for 14-3-3 protein, a negative regulator of Raf. Similarly, phosphorylation of BAD or the forkhead transcription factor FKHRL1 by Akt also promotes binding of 14-3-3 protein. In all
three instances, phosphorylation by Akt inactivates the function of its substrate. Cross talk between the Raf-MEK-ERK and the PI 3-kinase-Akt pathways,
mediated by direct interaction of Akt with and its phosphorylation of Raf, may switch the biological response from growth arrest to proliferation, as shown for
MCF-7 cells, and may also modulate senescence or differentiation as shown for myoblast differentiation, depending on the cellular system (Zimmermann, 1999).
NGF is a target-derived growth factor for developing sympathetic neurons. Application of NGF exclusively to distal axons of sympathetic
neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of
neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K
signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement of PI3-K signaling in distal axons for survival may be
explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles
within spatially distinct cellular locales during retrograde NGF signaling (Kuruvilla, 2000).
Dissociated sympathetic neurons obtained from newborn rat superior cervical ganglia and grown in compartmentalized cultures were to assess the subcellular distribution and state of activation of PI3-K and its downstream effector Akt (protein kinase B). Neurons were maintained under conditions in which cell bodies and proximal axons (hereafter referred to as the cell body compartment) were exposed to medium containing a neutralizing antibody directed against NGF (alpha-NGF), while distal axons, which are >1 mm away from cell bodies, were exposed to medium containing NGF. These conditions resemble in vivo conditions in which neurons are maintained by NGF acting exclusively on distal axons (Kuruvilla, 2000).
It was asked whether binding of NGF to receptors exclusively on distal axons regulates the activities of PI3-K and Akt in distal axons and/or cell bodies. For these experiments, NGF was removed from medium bathing distal axons for 24 hr. Then, distal axons were exposed to the same medium (control) or medium containing NGF for various times. The activation states of TrkA and Akt were assessed in extracts prepared from cell body and distal axon compartments by immunoblotting using antibodies that recognize the activated, phosphorylated forms of these proteins. P-Trk (Y490) antibodies recognize TrkA when phosphorylated on Tyr-490, which is the Shc recognition site. P-Akt antibodies recognize Akt when phosphorylated on Ser-473, which is necessary for its catalytic activity. Application of NGF to distal axons results in increased levels of P-TrkA (Y490) and P-Akt within distal axons, which are maximal after 20 min. Increases in both P-TrkA (Y490) and P-Akt are also detected in cell bodies but with slower kinetics. A small but reproducible increase in both P-TrkA (Y490) and P-Akt is detected in extracts of cell bodies within 20 min, and a more robust increase is seen at 8 hr. The appearance of P-TrkA (Y490) and P-Akt in both distal axons and cell bodies is coincident with the appearance of PI3-K activity associated with phosphotyrosine immunoprecipitates. Additionally, withdrawal of NGF from distal axons of neurons, which had been grown with medium containing a high concentration of NGF (100 ng/ml) on distal axons and alpha-NGF on cell bodies, leads to a decrease in the levels of both P-TrkA (Y490) and P-Akt in distal axons and in cell bodies. Thus, NGF acting on TrkA receptors on distal axons regulates the phosphorylation/activation of TrkA, PI3-K, and Akt both locally within distal axons and retrogradely to proximal axons and cell bodies of sympathetic neurons (Kuruvilla, 2000).
These results support the idea that PI3-K signaling within both cell bodies and distal axons is necessary for survival of neurons supported by NGF acting on distal
axons. Moreover, the requirement of PI3-K signaling in distal axons is more apparent when a submaximal concentration of NGF is used to support
survival. How does PI3-K signaling within distal axons contribute to survival? It was found that PI3-K activity in distal axons controls retrograde NGF transport
and retrograde signaling, which may be critical for survival. Complete inhibition of PI3-K in distal axons, as assessed by levels of P-Akt, attenuates retrograde
transport of NGF by ~80% in two compartment chambers and 65% in three compartment chambers. Thus, there is a small but significant amount of retrograde
transport that occurs in a PI3-K-independent manner. These observations may account for the finding that inhibition of PI3-K in distal axons has more dire
consequences for neurons supported by 0.5 ng/ml NGF acting on distal axons than for those supported by 50 ng/ml NGF acting on distal axons. Neurons
grown in a low, submaximal concentration of NGF are more vulnerable than neurons supported by a high concentration of NGF to a 65%-80% reduction in
retrograde signaling (Kuruvilla, 2000).
The precise role of PI3-K signaling in distal axons for ligand-dependent internalization, retrograde transport, and retrograde signaling is not clear. It is possible
that products of the PI3-K catalyzed reaction are critical for the ligand-dependent production of clathrin-coated pits, into which NGF and TrkA are initially
internalized. In support of this idea, there is an essential role for the pleckstrin homology (PH) domain of the
GTPase dynamin for receptor-mediated endocytosis. Further, dynamin is required for retrograde
transport of NGF in sympathetic neurons. Since the dynamin PH domain binds to phosphoinositide products of the
PI3-K-catalyzed reaction, PI3-K activity associated with TrkA may be critical for recruitment of dynamin to regions of the plasma
membrane destined to invaginate to form NGF/TrkA-containing clathrin-coated signaling organelles. Similarly, AP-2, which is involved in clathrin coat formation
and vesicle sorting at the plasma membrane, contains an amino-terminal phosphoinositide binding domain that is required for its targeting to the plasma
membrane. Thus, it is tempting to speculate that PI3-K signaling in distal axons is needed for survival because this TrkA effector
controls membrane recruitment of key regulators of NGF/TrkA endocytosis and retrograde TrkA signaling (Kuruvilla, 2000).
If PI3-K in distal axons is required for retrograde signaling, what is the role of PI3-K in cell bodies in neurons supported by NGF acting exclusively on distal
axons? Inhibition of PI3-K in cell bodies leads to near complete apoptosis of neurons within 48 hr, but inhibition of PI3-K exclusively in cell bodies does not affect
retrograde transport of NGF. Under these conditions, P-Akt in cell bodies is completely blocked, but levels of P-Akt in distal axons are unaffected. These
observations indicate that PI3-K and Akt signaling in distal axons alone cannot support neuronal survival. Since constitutively active PI3-K and Akt can support
survival of sympathetic neurons, it is speculated that PI3-K signaling in cell bodies is necessary for survival because it supports Akt signaling and phosphorylation
of Akt substrates that mediate the prosurvival effects of PI3-K. Indeed, it seems likely that many of the substrates of Akt function, at least in part, within cell
bodies. Substrates of Akt include BAD, caspase-9, IKK, the transcription factor forkhead, and, possibly, CREB. By extension, these data support the idea that phosphorylation of Akt substrates within distal axons cannot support neuronal survival. This may be
because critical substrates of Akt are either not present in distal axons or that they are present in distal axons but cannot move in the phosphorylated forms from
distal axons to cell bodies to affect the apoptotic machinery. P-Akt itself does not move from distal axons to cell bodies to an appreciable
extent so the same is likely to be true for products of Akt-catalyzed phosphorylation reactions (Kuruvilla, 2000).
Growth factor signal transduction mechanisms in neurons are arguably more complex than in most other cell types due to the striking morphological
specializations of neurons. Most neurons have long axons that can extend centimeters or even one meter from their cell bodies, and target-derived growth factor
signals must be propagated over long distances to influence survival and gene expression within cell bodies. These retrograde signals must be integrated with
signals coming from dendrites and those emanating from the membrane of the cell body itself. The present study shows that the same NGF effector pathway, the
PI3-K pathway, can have different functions in distinct parts of the same neuron during long-range retrograde signaling. Interestingly, the activity of the PI3-K
signaling in distal axons indirectly regulates TrkA signaling pathways in cell bodies, including the PI3-K effector pathway. Thus, there exists interdependence of
TrkA effector pathways in distinct cellular locales whereby ligand-dependent TrkA effector signaling in one compartment, the distal axon, controls effector
signaling in another, the cell body (Kuruvilla, 2000).
Studies in Dictyostelium have shown that the p110-related phosphatidylinositol-3-kinases PI3K1 and PI3K2 are required for proper development, pinocytosis chemotaxis, and chemoattractant-mediated activation of PKB. Insights into the mechanism by which PI3K regulates chemotaxis derive from studies on PKB in mammalian leukocytes and Dictyostelium cells. PKB activation requires its translocation to the plasma membrane by binding of its PH domain to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 produced upon activation of PI3K, leading to PKB activation. In leukocytes and Dictyostelium cells, chemoattractants mediate PKB activation through a G-protein-coupled pathway that requires the activity of the respective PI3Ks. Chemoattractant stimulation of neutrophils and Dictyostelium cells results in a transient localization of a GFP fusion of the PH domains from the Dictyostelium and mammalian PKBs to the plasma membrane. When these cells are placed in a chemoattractant gradient, membrane localization of the PKB-PH-GFP fusion is restricted to the leading edge, as is the case for other PH-domain-containing proteins in Dictyostelium. In Dictyostelium, translocation of the PKB-PH domain GFP fusion is PI3K-dependent. PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, a structural homolog of mammalian PAKs (p21-activated kinase) that is required for myosin II assembly. PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis (Chung, 2001).
The phosphatidylinositol 3-kinase signaling pathway has inherent
oncogenic potential. It is up-regulated in diverse human cancers by either a
gain of function in PI3K itself or in its downstream target Akt, or by a loss of
function in the negative regulator PTEN. However, the complete consequences of
this up-regulation are not known. Insulin and epidermal growth
factor or an inactivating mutation in the tumor suppressor PTEN specifically
increase the protein levels of hypoxia-inducible factor (HIF) 1alpha but not of
HIF-1beta in human cancer cell lines. This specific elevation of HIF-1alpha
protein expression requires PI3K signaling. In the prostate carcinoma-derived
cell lines PC-3 and DU145, insulin- and epidermal growth factor-induced
expression of HIF-1alpha is inhibited by the PI3K-specific inhibitors LY294002
and wortmannin in a dose-dependent manner. HIF-1beta expression is not affected
by these inhibitors. Introduction of wild-type PTEN into the PTEN-negative PC-3
cell line specifically inhibits the expression of an HIF-1alpha but not that of
HIF-1beta. In contrast to the HIF-1alpha protein, the level of HIF-1alpha mRNA
is not significantly affected by PI3K signaling. Vascular endothelial growth
factor reporter gene activity is induced by insulin in PC-3 cells and is
inhibited by the PI3K inhibitor LY294002 and by the coexpression of a HIF-1
dominant negative construct. Vascular endothelial growth factor reporter gene
activity is also inhibited by expression of a dominant negative PI3K construct
and by the tumor suppressor PTEN (Jiang, 2001).
The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. Cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice has revealed that the cardiac hypertrophy and the contractility defects can be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility (Crackower, 2002).
Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27(kip1), thereby promoting CDK2 activity and cellular proliferation. These results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function (Dan, 2002).
Loss of PTEN causes unregulated activation of downstream components of phosphatidylinositol 3-kinase (PI3K) signaling, including PDK1, and disrupts normal nervous system development and homeostasis. This study tested the contribution of Pdk1 to the abnormalities induced by Pten deletion in the brain. Conditional deletion of Pdk1 caused microcephaly. Combined deletion of Pdk1 and Pten rescued hypertrophy, but not migration defects of Pten-deficient neurons. Pdk1 inactivation induced strikingly different effects on the regulation of phosphorylated Akt in glia versus neurons. These results show Pdk1-dependent and Pdk1-independent abnormalities in Pten-deficient brains, and demonstrate cell type specific differences in feedback regulation of the ubiquitous PI3K pathway (Chalhoub, 2009).
Cell-to-cell variability in populations has been widely observed in mammalian cells. This heterogeneity can result from random stochastic events or can be deliberately maintained through regulatory processes. In the latter case, heterogeneity should confer a selective advantage that benefits the entire population. Using multicolor flow cytometry, this study has uncovered robust heterogeneity in phosphoinositide 3-kinase (PI3K) activity in MCF10A cell populations, which had been previously masked by techniques that only measure population averages. AKT activity is bimodal in response to EGF stimulation and correlates with PI3K protein level, such that only cells with high PI3K protein can activate AKT. It was further shown that heterogeneity in PI3K protein levels is invariably maintained in cell populations through a degradation/resynthesis cycle that can be regulated by cell density. Given that the PI3K pathway is one of the most frequently upregulated pathways in cancer, it is proposed that heterogeneity in PI3K activity is beneficial to normal tissues by restricting PI3K activation to only a subset of cells. This may serve to protect the population as a whole from overactivating the pathway, which can lead to cellular senescence or cancer. Consistent with this, it was show that oncogenic mutations in p110α (H1047R and E545K) partially evade this negative regulation, resulting in increased AKT activity in the population (Yuan, 2011).
Although cytoplasmic PI3Kinase (PI3K) is well characterized, regulation of nuclear PI3K has been obscure. A novel protein, PIKE (PI3Kinase Enhancer), interacts
with nuclear PI3K to stimulate its lipid kinase activity. PIKE encodes a 753 amino acid nuclear GTPase. Dominant-negative PIKE prevents the NGF enhancement
of PI3K and upregulation of cyclin D1. NGF treatment also leads to PIKE interactions with 4.1N, which has translocated to the nucleus, fitting with the initial
identification of PIKE based on its binding 4.1N in a yeast two-hybrid screen.
Protein 4.1N is a neuronal selective isoform of the erythrocyte membrane cytoskeleton protein 4.1R. Protein 4.1N binds the
nuclear mitotic apparatus protein (NuMA), a nonhistone nuclear protein that leaves the nucleus at mitosis and is associated with poles of the mitotic spindle. 4.1N mediates the antimitotic antiproliferative actions of
NGF. Overexpression of 4.1N abolishes PIKE effects on PI3K. Activation of nuclear PI3K
by PIKE is inhibited by the NGF-stimulated 4.1N translocation to the nucleus. Thus, PIKE physiologically modulates the activation by NGF of nuclear PI3K (Ye, 2000).
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