Phosphotidylinositol 3 kinase 92E
Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then
function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. A novel assay has been established in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo, and has been termed
TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange
factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK)
rescues yeast growth at the non-permissive temperature of 37 degrees C. Using this assay, several amino acids in the beta1-beta2 region of PH
domains have been identified that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and a structural model has been proposed for how these PH domains
might bind PI3K products with high affinity. From these data, a consensus sequence has been derived that predicts high-affinity binding to PtdIns(3,4)P2 and/or
PtdIns(3,4,5)P3. Several new PH domain-containing proteins have been identified that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this
assay to screen for novel cDNAs that rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K (Isakoff, 1999).
Gab1 has structural similarities to Drosophila Dos (Daughter of sevenless); Dos is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and
Dos have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are
phosphorylated. Gab1 is tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon
(IFN-alpha), and IFN-gamma. Upon the stimulation of IL-6 or IL-3, Gab1 is found to form a complex with phosphatidylinositol PI3 kinase and SHP-2, a
homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, reveals that neither tyrosine residues of gp130 nor its
carboxy terminus is required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhances gp130-dependent mitogen-activated protein (MAP) kinase
ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogates the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2
activation. Furthermore, ERK2 activation is inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations
suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras
are involved in Gab1-mediated ERK activation (Takahashi-Tezuka, 1998).
GAP1(m) is a member of the GAP1 family of Ras GTPase-activating proteins (GAPs). In vitro, it has been shown to bind inositol 1,3,4,5-tetrakisphosphate
(IP4), the water-soluble inositol head group of the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). This suggests
that GAP1(m) might function as a PIP3 receptor in vivo. This study, using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing a
chimera of green fluorescent protein fused to GAP1(m) [GFP-GAP1(m)] shows that epidermal growth factor (EGF) induces a rapid (less than 60 seconds)
recruitment of GFP-GAP1(m) from the cytosol to the plasma membrane. This recruitment requires a functional GAP1(m) pleckstrin homology (PH) domain,
because a specific point mutation (R629C) in the PH domain that inhibits IP4 binding in vitro totally blocks EGF-induced GAP1(m) translocation. Furthermore,
the membrane translocation is dependent on PI 3-kinase, and the time course of translocation parallels the rate by which EGF stimulates the generation of plasma
membrane PIP3. Significantly, the PIP3-induced recruitment of GAP1(m) does not appear to result in any detectable enhancement in its basal Ras GAP activity.
From these results, it is concluded that GAP1(m) binds PIP3 in vivo: it is recruited to the plasma membrane, but does not appear to be activated, following
agonist stimulation of PI 3-kinase (Lockyer, 1999).
The activation status of the guanosine triphosphate (GTP)-binding protein Ras is dictated by the relative intensities of two opposing reactions: the formation of active Ras-GTP complexes, promoted by guanine-nucleotide exchange factors (GEFs), and their conversion to inactive Ras-GDP as a result of the deactivating action of GTPase-activating proteins (GAPs). The relevance of phosphoinositide 3-kinase (PI 3-kinase) to these processes is still unclear. The regulation of Ras activation by PI 3-kinase has been investigated in the myelomonocytic U937 cell line. These cells exhibit basal levels of Ras-GTP, which are suppressed by two PI 3-kinase inhibitors and a dominant-negative PI 3-kinase. In addition, PI 3-kinase inhibition aborts Ras activation by all stimuli tested, including fetal calf serum (FCS) and phorbol 12-myristate 13-acetate (TPA). Significantly, TPA does not activate PI 3-kinase in U937 cells, indicating that PI 3-kinase has a permissive rather than an intermediary role in Ras activation. Investigation of the mechanism of PI 3-kinase action reveals that inhibition of PI 3-kinase does not affect nucleotide exchange on Ras but abrogates Ras-GTP accumulation through an increase in GAP activity. These findings establish blockage of GAP action as the mechanism underlying a permissive function of PI 3-kinase in Ras activation (Rubio, 2000)
The increase in GAP activity induced by PI 3-kinase inhibitors indicates that resting levels of the lipids produced in the plasma membrane by PI 3-kinase inhibit the action of GAP proteins on Ras. Wortmannin pretreatment of U937 cells does not alter GAP activity as assayed from cell lysates. This suggests that membrane integrity is important for PI3-kinase-mediated inhibition of GAPs. Considering that Ras can activate PI 3-kinase through a direct interaction with the p110 catalytic subunit, these data suggest the following scenario. Active Ras could activate PI 3-kinase to induce spatially restricted generation of 3-phosphoinositides. This would promote local downregulation of relevant GAP species and thus allow basal Ras activation (Rubio, 2000).
Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) has been proposed to act as a second messenger to recruit regulatory proteins
to the plasma membrane via their pleckstrin homology (PH) domains. The PH domain of Bruton's tyrosine kinase (Btk), which is
mutated in the human disease X-linked agammaglobulinemia, has been shown to interact with PI(3,4,5)P3 in vitro. In this study, a
fusion protein containing the PH domain of Btk and the enhanced green fluorescent protein (BtkPH-GFP) was constructed and utilized
to study the ability of this PH domain to interact with membrane inositol phospholipids inside living cells. The localization of
expressed BtkPH-GFP in quiescent NIH 3T3 cells was indistinguishable from that of GFP alone, both being cytosolic as assessed by
confocal microscopy. In NIH 3T3 cells coexpressing BtkPH-GFP and the epidermal growth factor receptor, activation of epidermal
growth factor or endogenous platelet-derived growth factor receptors causes a rapid
(<3 min) translocation of the cytosolic
fluorescence to ruffle-like membrane structures. This response is not observed in cells expressing GFP only, and is completely
inhibited by treatment with the PI 3-kinase inhibitors wortmannin and LY 292004. Membrane-targeted PI 3-kinase also causes
membrane localization of BtkPH-GFP that is slowly reversed by wortmannin. When the R28C mutation of the Btk PH domain,
which causes X-linked agammaglobulinemia, is introduced into the fluorescent construct, no translocation is observed after
stimulation. In contrast, the E41K mutation, which confers transforming activity to native Btk, causes significant membrane
localization of BtkPH-GFP with characteristics indicating its possible binding to PI(4,5)P2. This mutant, but not wild-type
BtkPH-GFP, interferes with agonist-induced PI(4,5)P2 hydrolysis in COS-7 cells. These results show in intact cells that the PH
domain of Btk binds selectively to 3-phosphorylated lipids after activation of PI 3-kinase enzymes and that losing such binding ability
or specificity results in gross abnormalities in the function of the enzyme. Therefore, the interaction with PI(3,4,5)P3 is likely to be an
important determinant of the physiological regulation of Btk and can be utilized to visualize the dynamics and spatiotemporal
organization of changes in this phospholipid in living cells (Varnai, 1999).
Both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin-mediated cell-cell adhesion in epithelial
Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring
E-cadherin-mediated cell-cell adhesion. The Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin-mediated cell-cell adhesion. On different collagens, however, expression of
Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens
junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase-PAK
protein, is similar on fibronectin or collagen I, suggesting, rather, that the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular
responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin-mediated cell-cell
adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac (Sander, 1998).
Bile acid transport and secretion in hepatocytes require phosphatidylinositol (PI) 3-kinase-dependent recruitment of ATP-dependent
transporters to the bile canalicular membrane and are accompanied by increased canalicular PI 3-kinase activity. The
lipid products of PI 3-kinase also regulate ATP-dependent transport of taurocholate and dinitrophenyl-glutathione directly in canalicular
membranes. ATP-dependent transport of taurocholate and dinitrophenyl-glutathione in isolated canalicular vesicles from rat liver is
reduced 50%-70% by PI 3-kinase inhibitors, wortmannin, and LY294002, at concentrations that are specific for Type I PI 3-kinase.
Inhibition is reversed by addition of lipid products of PI 3-kinase (PI 3,4-bisphosphate and, to a lesser extent, PI 3-phosphate and
PI 3,4,5-trisphosphate) but not by PI 4, 5-bisphosphate. A membrane-permeant synthetic 10-mer peptide that binds
polyphosphoinositides and leads to activation of PI 3-kinase in macrophages doubles PI 3-kinase activity in canalicular membrane
vesicles and enhances taurocholate and dinitrophenyl-glutathione transport in canalicular membrane vesicles above maximal
ATP-dependent transport. The effect of the peptide is blocked by wortmannin and LY294002. PI 3-kinase activity is also
necessary for function of the transporters in vivo. ATP-dependent transport of taurocholate and PI 3-kinase activity are reduced in
canalicular membrane vesicles isolated from rat liver that have been perfused with taurocholate and wortmannin. PI 3,4-bisphosphate
enhances ATP-dependent transport of taurocholate in these vesicles above control levels.
These results indicate that PI 3-kinase lipid
products are necessary in vivo and in vitro for maximal ATP-dependent transport of bile acid and nonbile acid organic anions across
the canalicular membrane. These results demonstrate regulation of membrane ATP binding cassette transporters by PI 3-kinase lipid
products (Misra, 1999).
Recognition of phosphatidylinositol 3-phosphate [PtdIns(3)P] is crucial for a broad range of cellular signaling and membrane trafficking events regulated by
phosphoinositide (PI) 3-kinases. PtdIns(3)P binding by the FYVE domain of human early endosome autoantigen 1 (EEA1), a protein implicated in endosome fusion,
involves two beta hairpins and an alpha helix. Unlike other domains that bind PtdIns derivatives, the FYVE domain
requires two zinc ions coordinated by eight cysteines. FYVE domain binding to PtdIns(3)P is abolished by cysteine mutations or zinc
chelation. FYVE domain family members share ~40% sequence identity and are defined
by an RRHHCRXCG motif (where X is any residue) required for PtdIns(3)P interaction and a conserved region N-terminal to the first
cysteine. Specific amino acids, including those of the FYVE domain's conserved RRHHCRQCGNIF motif,
contact soluble and micelle-embedded lipid and provide specificity for PtdIns(3)P over PtdIns(5)P and PtdIns, as shown by heteronuclear magnetic resonance
spectroscopy. Although the FYVE domain relies on a zinc-binding motif reminiscent of RING fingers, it is distinguished by novel structural features and its
PtdIns(3)P-binding site (Kutateladze, 1999).
The EEA1 FYVE domain binds both PtdIns(3)P and micelles and forms a functionally important dimer. In addition to PtdIns(3)P-binding residues, Lys-1347 is implicated in lipid recognition by NMR. The EEA1 residue Arg-1374 (which is equivalent to Arg-193 of another FYVE protein Vps27p) is
influenced similarly by PtdIns(3)P and PtdIns(5)P based on chemical shift changes and therefore is not expected to determine specificity for the 3-phosphate. EEA1's
hydrophobic element that is predicted to insert into membrane is more extensive, while electrostatic membrane interactions suggested for Vps27p residue Lys-181 are
not supported by micelle-specific chemical shift changes in the corresponding EEA1 residue, Lys-1362. The direct identification of specific lipid-binding residues by
NMR and mutational experiments provide opportunities to study the mechanism of PtdIns(3)P and membrane binding in order to gain further insight into the complex
role of FYVE domains in membrane trafficking and signal transduction (Kutateladze, 1999).
Protein kinase B (PKB), and the p70 and p90 ribosomal S6 kinases (p70 S6 kinase and p90 Rsk, respectively), are activated by phosphorylation of two residues, one in the 'T-loop' of the kinase domain and, the other, in the hydrophobic motif carboxy terminal to the kinase domain. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds with high affinity to the PI 3-kinase lipid product phosphatidylinositol-3,4,5-trisphosphate, activates many AGC kinases in vitro by phosphorylating the T-loop residue, but whether PDK1 also phosphorylates the hydrophobic motif and whether all other AGC kinases are substrates for PDK1 is unknown. Mouse embryonic stem (ES) cells in which both copies of the PDK1 gene were disrupted are viable. In PDK1-/- ES cells, PKB, p70 S6 kinase and p90 Rsk (see Drosophila S6kII) are not activated by stimuli that induced strong activation in PDK1+/+ cells. Other AGC kinases – namely, protein kinase A (PKA), the mitogen- and stress-activated protein kinase 1 (MSK1) and the AMP-activated protein kinase (AMPK) – have normal activity or are activated normally in PDK1-/- cells. The insulin-like growth factor 1 (IGF1) induces PKB phosphorylation at its hydrophobic motif, but not at its T-loop residue, in PDK1-/- cells. IGF1 does not induce phosphorylation of p70 S6 kinase at its hydrophobic motif in PDK1-/- cells. It is concluded PDK1 mediates activation of PKB, p70 S6 kinase and p90 Rsk in vivo, but is not rate-limiting for activation of PKA, MSK1 and AMPK. Another kinase phosphorylates PKB at its hydrophobic motif in PDK1-/- cells. PDK1 phosphorylates the hydrophobic motif of p70 S6 kinase either directly or by activation of another kinase (Williams, 2000).
Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, it has been found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal
adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincides with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remain localized to focal adhesions, suggesting that activation of PI 3-kinase induces a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3), a lipid product of PI 3-kinase, is sufficient to induce restructuring of the focal adhesion plaque. PtdIns (3,4,5)-P3 binds to alpha-actinin in PDGF-treated cells. Activation of PI 3-kinase by PDGF induces a decrease in the association of alpha-actinin with the integrin beta subunit, and PtdIns (3,4,5)-P3 can disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P3, has important implications for the regulation of cellular adhesive strength and motility (Greenwood, 2000).
Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], play important roles in signaling processes crucial for cell movement, differentiation and survival.
A 32kDa PtdIns(3,4,5)P3-binding protein from porcine leukocytes has been isolated. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. The subcellular distribution of DAPP1 in response to cell stimulation has been characterized. When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocates from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation is dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 enters the cell in vesicles. Similar responses are seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalization studies in PAE cells suggest entry of DAPP1 by
endocytosis in a population of early endosomes containing internalized PDGF-ß receptors. DAPP1 also undergoes PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event is involved in the vesicular response (Anderson, 2000).
Rel/NF-kappaB transcription factors regulate the division and survival of B lymphocytes. B cells lacking NF-kappaB1 and c-Rel fail to increase in size upon mitogenic stimulation due to a reduction in induced c-myc expression. Mitogen-induced B cell growth, although not markedly impaired by FRAP/mTOR or MEK inhibitors, requires phosphatidylinositol 3-kinase (PI3K) activity. Inhibition of PI3K-dependent growth coincides with a block in the nuclear import of NF-kappaB1/c-Rel dimers and a failure to upregulate c-myc. In addition, PI3K has been shown to be necessary for a transcription-independent increase in c-Myc protein levels that accompanies mitogenic activation. Collectively, these findings establish a role for Rel/NF-kappaB signaling in the mitogen-induced growth of mammalian cells; such growth in B lymphocytes requires a PI3K/c-myc-dependent pathway (Grumont, 2003).
Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. A tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of GSK-3ß. These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). These results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway (Zhou. 2004).
Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. This study shows that insulin stimulates the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increases in PTEN-overexpressing cells and decreased in PTEN-deficient cells. Insulin induced the accumulation of SSH1L and active Akt, together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulates in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolishes insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin (Nishita, 2004).
Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger
domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the
physiological role of Hrs, mice were generated carrying a null mutation of the gene. Hrs homozygous mutant embryos develop with their ventral
region outside of the yolk sac, have two independent bilateral heart tubes (cardia bifida), lack a foregut, and die around embryonic day 11 (E11).
These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis is detected in the
ventral region of mutant embryos within the definitive endoderm, suggesting an important role for this germ layer in ventral folding morphogenesis.
Abnormally enlarged early endosomes are detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in
vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs is disrupted in cells treated with
wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking (Komada, 1999).
How a neuron becomes polarized remains an as yet unanswered question. Selection of the future axon among neurites of a cultured hippocampal neuron requires the activity of growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase (PI 3-kinase), as well as atypical protein kinase C (aPKC). The PI 3-kinase activity, highly localized to the tip of the newly specified axon of stage 3 neurons, is essential for the proper subcellular localization of mPar3, the mammalian homolog of C. elegans polarity protein Par3. Polarized distribution of not only mPar3 but also mPar6 is important for axon formation; ectopic expression of mPar6 or mPar3, or just the N terminus of mPar3, leaves neurons with no axon specified. Thus, neuronal polarity is likely to be controlled by the mPar3/mPar6/aPKC complex and the PI 3-kinase signaling pathway, both serving evolutionarily conserved roles in specifying cell polarity (Shi, 2003).
Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Exogenous PtdIns(3,4,5)P3 was ectopically inserted into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface (Gassama-Diagne, 2006).
Cell polarity is crucial for directed migration. This study shows that phosphoinositide 3-kinase (PI(3)K) mediates neutrophil migration in vivo by differentially regulating cell protrusion and polarity. The dynamics of PI(3)K products PI(3,4,5)P(3)-PI(3,4)P(2) during neutrophil migration were visualized in living zebrafish, revealing that PI(3)K activation at the leading edge is critical for neutrophil motility in intact tissues. A genetically encoded photoactivatable Rac was used to demonstrate that localized activation of Rac is sufficient to direct migration with precise temporal and spatial control in vivo. Similar stimulation of PI(3)K-inhibited cells did not direct migration. Localized Rac activation rescued membrane protrusion but not anteroposterior polarization of F-actin dynamics of PI(3)K-inhibited cells. Uncoupling Rac-mediated protrusion and polarization suggests a paradigm of two-tiered PI(3)K-mediated regulation of cell motility. This work provides new insight into how cell signaling at the front and back of the cell is coordinated during polarized cell migration in intact tissues within a multicellular organism (Yoo, 2010).
Self-renewal of embryonic stem cells (ESCs) is essential for maintenance of pluripotency, which is defined as the ability to differentiate into any specialised cell type comprising the adult organism. Understanding the mechanisms that regulate ESC self-renewal and proliferation is required before ESCs can fulfil their potential in regenerative therapies, and murine ESCs (mESCs) have been widely used as a model. Members of the class-IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses, including cell migration, proliferation and survival. PI3Ks have been reported to regulate both proliferation and self-renewal of mESCs. This study investigated the contribution of specific class-IA PI3K isoforms to the regulation of mESC fate using small-molecule inhibitors with selectivity for particular class-IA PI3K catalytic isoforms, and siRNA-mediated knockdown. Pharmacological inhibition or knockdown of p110beta promoted mESC differentiation, accompanied by a decrease in expression of Nanog. By comparison, pharmacological inhibition or siRNA-mediated knockdown of p110alpha had no effect on mESC self-renewal per se, but instead appeared to reduce proliferation, which was accompanied by inhibition of leukaemia inhibitory factor (LIF) and insulin-induced PI3K signalling. These results suggest that PI3Ks contribute to the regulation of both mESC pluripotency and proliferation by differential coupling to selected p110 catalytic isoforms (Kingham, 2009).
Myogenesis in vertebrate myocytes is promoted by activation of the phosphatidyl-inositol 3'-kinase (PI3 kinase) pathway and inhibited by fibroblast growth factor (FGF) signaling. Hyperactivation of the Caenorhabditis elegans FGF receptor, EGL-15, similarly inhibits the differentiation of the hermaphrodite sex muscles. Activation of the PI3 kinase signaling pathway can partially suppress this differentiation defect, mimicking the antagonistic relationship between these two pathways known to influence vertebrate myogenesis. When ectopically expressed in body wall muscle precursor cells, hyperactivated EGL-15 can also interfere with the proper development of the body wall musculature. Hyperactivation of EGL-15 has also revealed additional effects on a number of fundamental processes within the postembryonic muscle lineage, such as cell division polarity. These studies provide important in vivo insights into the contribution of FGF signaling events to myogenesis (Sasson, 2004).
The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. This study shows that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. It is concluded that mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance (Chang, 2006).
Triggering of the macrophage cell line RAW 264.7 with LPS promotes a transient activation of phosphatidylinositol 3-kinase
(PI3-kinase). Incubation of activated macrophages with wortmannin and LY294002, two inhibitors of PI3-kinase, increased the
amount of inducible nitric oxide synthase (iNOS) and the synthesis of nitric oxide. Treatment with wortmannin promotes a prolonged
activation of NF-kappaB in LPS-treated cells as well as an increase in the promoter activity of the iNOS gene as deduced from
transfection experiments using a 1.7-kb fragment of the 5' flanking region of the iNOS gene. Cotransfection of cells with a
catalytically active p110 subunit of PI3-kinase impairs the responsiveness of the iNOS promoter to LPS stimulation, whereas
transfection with a kinase-deficient mutant of p110 maintains the up-regulation in response to wortmannin. These results indicate that
PI3-kinase plays a negative role in the process of macrophage activation and suggest that this enzyme might participate in the
mechanism of action of antiinflammatory cytokines (Diaz-Guerra, 1999).
Maturation of mouse oocytes is accompanied by an increase in sensitivity to inositol 1,4,5-trisphosphate (IP3)-mediated release of
intracellular calcium. To test the hypothesis that the maturation-associated 1.5- to 2.0-fold increase in the mass of the type 1 IP3 receptor
(IP3R-1) confers this increase in IP3 sensitivity, RNA interference was employed to prevent this change in IP3R-1 protein level.
Microinjection into germinal vesicle (GV)-intact oocytes of dsRNA corresponding to the IP3R-1 sequence results in a >90% reduction
in the amount of maternal IP3R-1 mRNA and prevents the maturation-associated increase in the mass of the IP3R-1 protein. These injected
oocytes mature to metaphase II: there was no effect on the maturation-associated increases in p34cdc2/cyclin B kinase and MAP kinase
activities or the global pattern of protein synthesis. IP3-induced cortical granule exocytosis is significantly decreased in these eggs when
compared with controls previously injected with enhanced green fluorescent protein (EGFP) dsRNA. Following insemination, the IP3R-1
dsRNA-injected eggs display significantly fewer Ca2+ transients than controls, and the duration of the first Ca2+ transient is about half
that of controls. These results support the hypothesis that the maturation-associated increase in the mass of IP3R-1 confers the increase in
IP3-sensitivity that is observed following oocyte maturation and is necessary for the proper Ca2+ oscillatory pattern following insemination (Xu, 2003).
The role of FGF signaling in early epithelial differentiation was investigated in
ES (embryonic stem) cell derived embryoid bodies. A dominant negative fibroblast growth factor receptor (FGFR) mutation was created by stably introducing into ES cells an Fgfr2 cDNA, truncated in its enzymatic domains. These cells failed to differentiate into cystic embryoid bodies. No epithelial differentiation and cavitation morphogenesis could be observed in the mutant, although its rate of cell proliferation remained unchanged. This phenotype was associated with a significant decrease in the activation of Akt/PKB and PLCgamma-1, as compared to the wild type, while the activation of MAPK/Erk was less affected. Requirement for PI 3-kinase signaling in embryoid body differentiation was demonstrated by specific inhibitors. Akt/PKB activation was abrogated by wortmannin in short-term experiments. In long-term cultures Ly294002 inhibited the differentiation of ES cells into embryoid bodies. These data demonstrate that for early epithelial differentiation FGF signaling is required through the PI 3-kinase-Akt/ PKB pathway (Chen, 2000).
Vertebrate TOP mRNAs contain an oligopyrimidine tract at their 5' termini (5'TOP) and encode components of the translational machinery. Previously it has been shown that they are subject to selective translational repression upon growth arrest and that their translational behavior correlates with the activity of S6K1. The translation of TOP mRNAs is rapidly repressed by amino acid withdrawal and this nutritional control depends strictly on the integrity of the 5'TOP motif. However, neither phosphorylation of ribosomal protein (rp) S6 nor activation of S6K1 per se is sufficient to relieve the translational repression of TOP mRNAs in amino acid-starved cells. Likewise, inhibition of S6K1 activity and rpS6 phosphorylation by overexpression of dominant-negative S6K1 mutants failed to suppress the translational activation of TOP mRNAs in amino acid-refed cells. Furthermore, TOP mRNAs are translationally regulated by amino acid sufficiency in embryonic stem cells lacking both alleles of the S6K1 gene. Inhibition of mTOR by rapamycin leads to fast and complete repression of S6K1, as judged by rpS6 phosphorylation, but to only partial and delayed repression of translational activation of TOP mRNAs. In contrast, interference in the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway by chemical or genetic manipulations rapidly and completely blocks the translational activation of TOP mRNAs. It appears, therefore, that translational regulation of TOP mRNAs, at least by amino acids, (1) is fully dependent on PI3-kinase, (2) is partially sensitive to rapamycin, and (3) requires neither S6K1 activity nor rpS6 phosphorylation (Tang, 2001).
Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. The ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Balpha (PKBalpha). Likewise, overexpression of constitutively active PI3-kinase or PKBalpha can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway (Stolovich, 2002).
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