Phosphotidylinositol 3 kinase 92E
Vascular endothelial growth factor (VEGF) receptor Flk-1/KDR in endothelial cells is activated during vasculogenesis and
angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing
signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway.
To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, its role was examined in human
umbilical vein endothelial cell (HUVEC) cycle progression. p85, the regulatory subunit of PI 3-kinase, is
constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promotes tyrosine autophosphorylation of
Flk-1/KDR and also induces phosphorylation of p85. This is followed by an increase in the PI 3-kinase activity, which is
sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induces a striking activation of MAP kinase in a time-dependent
manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to
show that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription
from the c-fos promoter and entry into the synthesis (S)-phase. These data demonstrate the importance of PI 3-kinase as a necessary
signaling component of VEGF-mediated cell cycle progression (Thakker, 1999).
Nerve growth factor induces differentiation and survival of rat PC12 pheochromocytoma cells. The activation of the erk cascade has
been implicated in transducing the multitude of signals induced by NGF. In order to explore the role of this signaling cascade in NGF
mediated survival, differentiation and proliferation, recombinant adenoviruses were generated that express the intermediates of the erk
cascade in their wild type, dominant negative and constitutively activated forms. Differentiation of PC12 cells has been shown to require
activity of the ras/erk pathway, whereas inhibition of this pathway has no effect on survival or proliferation. Constitutively active
forms of ras, raf and mek induce PC12 cell differentiation, while dominant interfering forms inhibite differentiation. Survival of
PC12 cells in serum-free medium does not require activity of the ras/erk pathway. Instead, PI3 Kinase signaling is necessary for
PC12 cell survival. Interestingly, constitutively activated versions of raf and mek are able to promote survival, but again this is
dependent on activation of PI3 Kinase. Therefore, at least two distinct signaling pathways are required in PC12 cells for mediation of
NGF functions (Kless, 1999).
Shp2, a protein tyrosine phosphatase possessing SH2 domains, is utilized in the intracellular signaling of various growth factors.
Shp2 is highly expressed in the CNS. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, which also
shows high levels of expression in the CNS, exerts neurotrophic and neuromodulatory effects in CNS neurons. How
BDNF utilizes Shp2 in its signaling pathway was examined in cultured cerebral cortical neurons. BDNF stimulates coprecipitation of
several tyrosine-phosphorylated proteins with anti-Shp2 antibody and Grb2 and phosphatidylinositol 3-kinase (PI3-K) are
coprecipitated with anti-Shp2 antibody in response to BDNF. In addition, both anti-Grb2 and anti-PI3-K antibodies coprecipitate
Shp2 in response to BDNF. The BDNF-stimulated coprecipitation of the tyrosine-phosphorylated proteins, Grb2, and PI3-K with
anti-Shp2 antibody is completely inhibited by K252a, an inhibitor of TrkB receptor tyrosine kinase. This BDNF-stimulated Shp2
signaling is markedly sustained as well as BDNF-induced phosphorylation of TrkB and mitogen-activated protein kinases. In PC12
cells stably expressing TrkB, both BDNF and nerve grow growth factor stimulate Shp2 signaling similar to that by BDNF in cultured
cortical neurons. These results indicated that Shp2 shows cross-talk with various signaling molecules including Grb2 and PI3-K in
BDNF-induced signaling and that Shp2 may be involved in the regulation of various actions of BDNF in CNS neurons (Yamada, 1999).
To determine how signals emanating from Trk transmit neurotrophin actions in primary neurons, an examination was made of the ability of TrkB mutated at defined effector binding sites
to promote sympathetic neuron survival or local axon growth. TrkB stimulates signaling proteins and induces survival and growth in a manner similar to TrkA. TrkB
mutated at the Shc binding site supports survival and growth poorly, relative to wild-type TrkB, whereas TrkB mutated at the PLC-gamma1 binding site
supports growth and survival well. TrkB-mediated neuronal survival is dependent on PI3-kinase and to a lesser extent MEK activity, while growth depends
upon both MEK and PI3-kinase activities. These results indicate that the TrkB-Shc site mediates both neuronal survival and axonal outgrowth by activating the
PI3-kinase and MEK signaling pathways (Atwall, 2000).
Thus, the Shc binding site in TrkB regulates the majority of sympathetic neuron survival and local axon growth. In the absence of this site, survival is reduced to 18% and growth reduced to ~30%, as compared to wild type TrkB-expressing neurons. No other single mutation in Trk significantly affects survival or growth in this manner. Mutation of the PLC-gamma1 binding site in combination with the Shc site completely suppresses survival but does not further reduce growth responses. It has been shown that in TrkBShc-/Shc- mice, the Shc site controls a small portion of BDNF-dependent survival (25% loss in the vestibular ganglion and no loss of BDNF-dependent nodose neurons), and the majority of NT-4-dependent survival in vivo. The Shc site is also essential for the survival in culture of nodose neurons in response to either BDNF or NT-4. Together with the data presented here, these results suggest that TrkB mediates survival largely via signaling through the Shc binding site. The discrepancy between in vivo and in vitro data for BDNF-dependent neuronal populations may arise because, in vivo, other growth factors may collaborate with BDNF-bound TrkB Shc- to compensate for the decrease in TrkB signaling through this site. Since BDNF has been shown to be a better ligand than NT-4 for TrkB Shc-, perhaps BDNF-dependent populations are better able to benefit from such compensation in vivo (Atwall, 2000 and references therein).
There have been few studies examining the role of Trk signaling pathways in the promotion and regulation of axonal growth. This issue has been examined using dissociated Xenopus spinal cultures from day-old embryos following blastomere injection with mRNA for wild-type or mutant Trk isoforms. One conclusion from that study was that PLC-gamma activation is essential for axon elongation. The results presented here using mammalian sympathetic neurons are very different. The TrkB PLC-gamma1- mutant, which is defective in activating PLC-gamma1 as assessed by tyrosine phosphorylation, displays no deficits in axon elongation in sympathetic neurons. Furthermore, growth promoted by TrkB encoding both Shc and PLC-gamma1 site mutations is no worse than by TrkB encoding only the Shc site mutation, further confirming that the PLC-gamma1 site plays no role in axon growth in these cultures. It has also been concluded that the PLC-gamma and PI3-kinase sites are both essential for local growth cone turning responses in Xenopus spinal neurons. Conversely, the Shc site is the most important for mediating local axon growth in sympathetic neurons. Thus, the data presented here, combining genetic and biochemical analysis, provides conclusive evidence that signaling through the Shc site mediates the majority of neurotrophin-dependent local
axon growth (Atwall, 2000 and references therein).
How does the Shc site mediate survival and axon outgrowth? Whereas mutation of the Shc site clearly eliminates Shc tyrosine phosphorylation, this mutation can effect other signaling processes such as FRS-2 binding to Trk. Thus, it cannot be conclusively said that Shc itself is required for these biological effects. However, the MEK/ERK and PI3-kinase/Akt pathways, both of which are likely dependent on Shc signaling through Ras, are essential for survival and local growth. TrkB mutated at the Shc site does retain a limited ability to stimulate axonal growth and induce ERK and Akt phosphorylation in response to BDNF. This may reflect the ability of Trk to use adaptor proteins other than Shc to stimulate these events. For example, TrkA mutated at the Shc site can activate ERK through the PLC-gamma1 site. TrkA also can stimulate ERK activity independently of the Shc site via rAPS and SH2-B, Akt via Gab1, and neuritogenesis in PC12 cells via SNT. Signaling proteins such as these whose activities are regulated independently of the Shc site may account for the residual 30% of axonal growth observed in neurons expressing TrkB Shc site mutants (Atwall, 2000 and references therein).
No report has described the signals involved in promoting local axon outgrowth. NGF promotes locally increased axonal growth in culture and increased target innervation in vivo. Attempts to address the intracellular signaling mechanisms that regulate such local responses have used systems where global or local affects on growth cannot be distinguished, and where survival and growth effects cannot be segregated. Here, using compartmented cultures, which overcome both of these confounding local issues, it was found that local TrkB-mediated axon elongation is dependent on both PI3-kinase and MEK activity, as activated via the Shc site. How do MEK and PI3-kinase promote local axonal growth? The MEK/ERK pathway is well known to phosphorylate microtubule-associated proteins (MAPs), including Tau, that regulate microtubule stability and control axonal elongation. ERKs also phosphorylate neurofilament proteins. Similarly, PI3-kinase regulates or associates with a number of cytoskeletal proteins, including actin, tubulin, and actin-regulating proteins. The studies presented here suggest that both the MEK/ERK and PI3-kinase pathways are ideally positioned to regulate local growth events ranging from the directionality of growth cone extension to axon elongation itself (Atwall, 2000 and references therein).
Studies reported here also demonstrate that the activities of PI3-kinase, and to a lesser extent MEK, are involved in regulating TrkB-induced sympathetic neuron survival. Evidence for the role of the PI3-kinase/Akt pathway in NGF-mediated survival of peripheral neurons is well documented. PI3-kinase/Akt regulates survival by inhibiting the activities of the cell death proteins Bad and the transcription factor Forkhead in cerebellar neurons and by suppressing the JNK/p53 cell death pathway in sympathetic neurons. In contrast to PI3-kinase/Akt, MEK activity is not important for regulating NGF-dependent peripheral neuron survival. However, MEK activity can mediate, to some extent, cell survival in neurotrophin-regulated neuronal systems, including the survival of cultured cerebellar granule neurons and retinal ganglion cells, and neuroprotection against camptothecin-induced death of cortical neurons and CA-induced death of sympathetic neurons. In addition, sympathetic neuronal survival promoted by activated Ras occurs partly through the MEK/ERK pathway. MEK, together with Akt, may regulate survival by activating the transcription factor CREB, which is a critical regulator of NGF-mediated neuronal survival of sympathetic neurons and of BDNF-mediated survival of cerebellar neurons (Atwall, 2000 and references therein).
In general, TrkA uses PI3-kinase activity to regulate cell survival, while TrkB uses both PI3-kinase and MEK. TrkB thus appears to function in a more 'flexible' manner, using multiple signaling pathways to control specific effects. TrkB may require such signaling flexibility to converge and synergize with many divergent survival cues that CNS neurons are exposed to. Whether such distinctions between TrkA and TrkB generalize to other neuronal responses such as growth, phenotypic modulation, or ongoing plasticity remains to be determined. Nevertheless, it may well be that TrkA may only have to make use of a single signaling pathway such as PI3-kinase to induce responses such as survival, suggesting that TrkA and TrkB are fundamentally different in their signaling capabilities (Atwall, 2000 and references therein).
Within minutes of its application, platelet-derived growth factor (PDGF) triggers cytoskeletal rearrangements and chemotaxis. These
events are at least in part due to the activation of phosphoinositide (PI) 3-kinase; there is good temporal correlation between these
events and the accumulation of 3-phosphorylated products of the kinase. Prolonged and continuous PDGF exposure results in S-phase
entry many hours after the initial burst of activity. Although early signals appear responsible for the early responses, they may not fully
account for later responses, such as cell-cycle progression.
An assessment was made of when PI 3-kinase products accumulate in
PDGF-stimulated cells. In addition to the previously identified early accumulation of products, a second, prolonged wave
of accumulation 3-7 hours after stimulation was detected. To determine the relative contribution of each phase to PDGF-dependent DNA synthesis,
an assay was developed in which synthetic 3-phosphorylated lipids were used to rescue DNA synthesis in cells expressing a
PDGF-receptor mutant. The lipids rescue DNA synthesis only when added 2-6 hours after PDGF. In addition, PI 3-kinase inhibitors
fail to block PDGF-dependent DNA synthesis if added during the first wave of PI 3-kinase activity, but adding them later, in G1
phase, prevents PDGF-dependent cell-cycle progression. It is concluded that PDGF induces distinct waves of PI 3-kinase activity.
The second wave is required for PDGF-dependent DNA synthesis, whereas the initial wave is not. One of the ways in which cells use
PI 3-kinase to mediate distinct cellular responses seems to be by regulating when products of PI 3-kinase accumulate (Jones, 1999).
Tumor necrosis factor (TNF) induces the phosphorylation of BAD at serine 136 in HeLa cells under conditions that are not cytotoxic.
BAD phosphorylation by TNF is dependent on phosphatidylinositide-3-OH kinase (PI3K) and is accompanied by the
translocation of BAD from the mitochondria to the cytosol. Blocking the phosphorylation of BAD and its translocation to the cytosol
with the PI3K inhibitor wortmannin activates caspase-3 and markedly potentiates the cytotoxicity of TNF. Transient transfection with
a PI3K dominant negative mutant or a dominant negative mutant of the serine-threonine kinase Akt, the downstream target of PI3K
and the enzyme that phosphorylates BAD, similarly potentiates the cytotoxicity of TNF. By contrast, transfection with a constitutively
active Akt mutant protects against the cytotoxicity of TNF in the presence of wortmannin. Phosphorylation of BAD prevents its
interaction with the antiapoptotic protein Bcl-XL. Transfection with a Bcl-XL expression vector protects against the cytotoxicity of
TNF in the presence of wortmannin. Discussed is the mechanism by which the inhibition of the phosphorylation of BAD is likely linked to the
induction of lethal mitochondrial damage in TNF-intoxicated cells (Pastorino, 1999).
E-cadherins are surface adhesion molecules localized at the level of adherens junctions, which play a major role in cell adhesiveness by
mediating calcium-dependent homophylic interactions at sites of cell-cell contacts. Recently, E-cadherins have been also implicated in a
number of biological processes, including cell growth and differentiation, cell recognition, and sorting during developmental
morphogenesis, as well as in aggregation-dependent cell survival. Since phosphatidylinositol (PI) 3-kinase and Akt play a critical role in
survival pathways in response to both growth factors and extracellular stimuli, these observations prompted an exploration of whether
E-cadherins could affect intracellular molecules regulating the activity of the PI 3-kinase/Akt signaling cascade. Using Madin-Darby
canine kidney cells as a model system, engagement of E-cadherins in homophylic calcium-dependent cell-cell
interactions has been shown to result in a rapid PI 3-kinase-dependent activation of Akt and the subsequent translocation of Akt to the nucleus.
Moreover, the activation of PI 3-kinase in response to cell-cell contact formation involves the phosphorylation of
PI 3-kinase in tyrosine residues, and the concomitant recruitment of PI 3-kinase to E-cadherin-containing protein complexes. These
findings indicate that E-cadherins can initiate outside-in signal transducing pathways that regulate the activity of PI 3-kinase and Akt,
thus providing a novel molecular mechanism whereby the interaction among neighboring cells and their adhesion status may ultimately
control the fate of epithelial cells (Pece, 1999).
Phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Evidence is provided that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. This study reports the following: (1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells; (2) reduction of hDlg expression levels by RNAi in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression; (3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells; (4) this interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit; (5) phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. It is concluded that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells (Laprise, 2004).
The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). Tyrosine phosphorylation of FRS2alpha leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2alpha:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway (Ong, 2001).
Classical cadherins mediate cell recognition and cohesion in many tissues of the
body. It is increasingly apparent that dynamic cadherin contacts play key roles
during morphogenesis and that a range of cell signals are activated as cells
form contacts with one another. It has been difficult, however, to determine
whether these signals represent direct downstream consequences of cadherin
ligation or are juxtacrine signals that are activated when cadherin adhesion
brings cell surfaces together but are not direct downstream targets of cadherin
signaling. In this study, a functional cadherin ligand (hE/Fc) has been used to
directly test whether E-cadherin ligation regulates phosphatidylinositol
3-kinase (PI 3-kinase) and Rac signaling. Homophilic cadherin
ligation recruits Rac to nascent adhesive contacts and specifically stimulates
Rac signaling. Adhesion to hE/Fc also recruits PI 3-kinase to the cadherin
complex, leading to the production of phosphatidylinositol 3,4,5-trisphosphate
in nascent cadherin contacts. Rac activation involves an early phase, which is
PI 3-kinase-independent, and a later amplification phase, which is inhibited by
wortmannin. PI 3-kinase and Rac activity are necessary for productive adhesive
contacts to form following initial homophilic ligation. It is concluded that
E-cadherin is a cellular receptor that is activated upon homophilic ligation to
signal through PI 3-kinase and Rac. It is proposed that a key function of these
cadherin-activated signals is to control adhesive contacts, probably via
regulation of the actin cytoskeleton, which ultimately serves to mediate
adhesive cell-cell recognition (Kovacs, 2002).
The signaling mechanisms utilized by the proinflammatory cytokine interleukin-1 (IL-1) to activate the transcription factors NFkappaB and activator
protein-1 (AP-1) are poorly defined. Evidence is presented that IL-1 not only stimulates a dramatic increase in phosphatidylinositol 3-kinase (PI
3-kinase) activity but also induces the physical interaction of its type I receptor with the p85 regulatory subunit of PI 3-kinase. Furthermore, two PI
3-kinase-specific inhibitors, wortmannin and a dominant-negative mutant of the p85 subunit, inhibit IL-1-induced activation of both NFkappaB and
AP-1. Transient transfection experiments indicated that whereas overexpression of PI 3-kinase may be sufficient to induce AP-1 and increase nuclear
c-Fos protein levels, PI 3-kinase may need to cooperate with other IL-1-inducible signals to fully activate NFkappaB-dependent gene expression. In this
regard, cotransfection studies suggested that PI 3-kinase may functionally interact with the recently-identified IL-1-receptor-associated kinase to activate
NFkappaB. These results thus indicate that PI 3-kinase is a novel signal transducer in IL-1 signaling and that it may differentially mediate the activation of NFkappaB and AP-1 (Reddy, 1997).
Phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear
factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. IL-1 is found to stimulate interaction
of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit.
Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression has no effect on the
IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In
contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit.
Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, it was found that overexpression of the p110
catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this
process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also
induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of
NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB (Sizemore, 1999).
Two distinct forms of short-term cytolysis have been described for CD8+ CTLs, the perforin/granzyme- and Fas ligand/Fas [CD95
ligand (CD95L)/CD95]-mediated pathways. However, the difference in signal transduction events leading to these cytolytic
mechanisms remains unclear. Wortmannin, an irreversible antagonist of phosphatidylinositol 3-kinase (PI3-K) activity, was used to
investigate the role of PI3-K in influenza-specific CD8+ CTL cytolytic effector function. The addition of wortmannin at
concentrations as low as 1 nM significantly inhibits both the Ag/MHC-induced cytolysis of CD95- target cells and serine esterase
release. In strong contrast, Wortmannin does not inhibit the Ag/MHC-induced CD95L expression or the CD95L/CD95-mediated cytolysis of
CD95+ targets. A combination of wortmannin and blocking mAb against CD95L inhibits the cytolysis of CD95+ targets, indicating
that the wortmannin-independent cytolysis is due to CD95L/CD95 mediated cytolysis. These findings suggest a differential role for
PI3-K in mediating cytolysis and, thus far, the earliest difference between perforin/granzyme- and CD95L/CD95-dependent cytolysis.
These data reinforce the idea of a TCR with modular signal transduction pathways that can be triggered or inhibited selectively, resulting
in differential effector function (Fuller, 1999).
Ca2+-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of
neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated
protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using
primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody, the ability of AMPA receptors to activate the MAPK cascade was addressed. In the presence of cyclothiazide, AMPA causes a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca2+ channels)
Ca2+-dependent activation of MAPK through MAPK kinase (MEK). This activation is blocked by GYKI 53655, a noncompetitive selective antagonist of
AMPA receptors. Probing the mechanism of this activation reveals an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a
pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca2+/calmodulin-dependent kinase II. Application of AMPA to rat cerebral cortical neurons has been shown to lead to a rapid increase in Ras activity and activation of MAPK. Ras-dependent activation of MAPK is usually associated with seven transmembrane receptors that couple to heterotrimeric G-proteins. AMPA activates ERK2 (p42) by causing a Ca2+-dependent association of G-protein betagamma subunits, probably Gi, with a Ras, Raf kinase, MEK complex. This novel
involvement of a heterotrimeric G-protein in ionotropic AMPA receptor signaling was examined. Striatal neurons were pretreated with pertussis toxin (PTX) or PBS vehicle for 24 hr before experiments with AMPA/cyclothiazide. PTX treatment abolishes AMPA receptor activation of MAPK, indicating a role for a Gi or Go-type G-protein
in the activation of MAPK by AMPA receptors in striatal neurons.
Similarly, kainate activates MAPK in a PI
3-kinase-dependent manner. AMPA receptor stimulation leads to a Ca2+-dependent phosphorylation of the nuclear transcription factor CREB, which can be prevented by
inhibitors of MEK or PI 3-kinase. These results indicate that Ca2+-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons
through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum (Perkinton, 1999).
Thus, although the specific protein-protein
interactions that lead to activation of the Ras-MAPK pathway by AMPA receptors are not currently known, it seems reasonable to propose that AMPA
receptor-evoked rises in cytosolic Ca2+ may trigger activation of PI 3-kinase: then, recruitment of the lipid kinase to the MAPK cascade may, as is the case with
seven-transmembrane Gi/Go-type G-protein linked receptors, be orchestrated by free G betagamma subunits. The specific exchange factors regulating
Ras activity after AMPA receptor stimulation also remain to be determined. An involvement of the neuron-specific guanine nucleotide exchange factor, Ras-GRF,
seems plausible because it has recently been demonstrated that Ras-GRF can be activated in response to increases in intracellular Ca2+ and/or free G-protein betagamma subunits that induce phosphorylation of Ras-GRF by as yet unknown
kinases. However, Ca2+/calmodulin-dependent activation of Ras-GRF does not appear to involve PTKs, thus, the results indicating that
tyrosine phosphorylation may be an important step in AMPA receptor activation of MAP kinase suggests that additional Ca2+-dependent routes to Ras may be
activated. It has been shown that CaM-KII can phosphorylate AMPA receptor subunits (Mammen et al., 1997), resulting in
enhanced receptor currents, and this has been implicated in the strengthening of postsynaptic
responses associated with synaptic plasticity. Selective inhibition of CaM-KII activity substantially reduces
AMPA/cyclothiazide-evoked activation of MAPK without altering Ca2+ influx through the receptor. These data indicate that CaM-KII can be a positive modulator
of AMPA receptor signaling but that in the presence of cyclothiazide the kinase probably regulates AMPA receptor-mediated MAPK activation at a point
downstream of Ca2+ entry (Perkinton, 1999 and references).
Inhibition of phosphatidylinositol (PI) 3-kinase severely attenuates the
activation of extracellular signal-regulated kinase (Erk)
following engagement of integrin/fibronectin receptors
and Raf is the critical target of PI 3-kinase
regulation. To investigate how PI 3-kinase regulates
Raf, sites on Raf1 required for regulation
by PI 3-kinase were examined and the mechanisms involved
in this regulation were explored. Serine 338 (Ser338), which 1s
critical for fibronectin stimulation of Raf1, is
phosphorylated in a PI 3-kinase-dependent manner
following engagement of fibronectin receptors. In
addition, fibronectin activation of a Raf1 mutant
containing a phospho-mimic mutation (S338D) is
independent of PI 3-kinase. Furthermore,
integrin-induced activation of the serine/threonine
kinase Pak-1, which has been shown to phosphorylate
Raf1 Ser338, is also dependent on PI 3-kinase activity,
and expression of a kinase-inactive Pak-1 mutant
blocks phosphorylation of Raf1 Ser338. These results
indicate that PI 3-kinase regulates phosphorylation of
Raf1 Ser338 through the serine/threonine kinase Pak.
Thus, phosphorylation of Raf1 Ser338 through
PI 3-kinase and Pak provides a co-stimulatory signal
which together with Ras leads to strong activation of
Raf1 kinase activity by integrins (Chaudhary, 2000).
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that
can phosphorylate phosphaditylinositides leading to the
cell type-specific regulation of intracellular protein kinases.
PI3Ks are involved in a wide variety of cellular events
including mitogenic signaling, regulation of growth
and survival, vesicular trafficking, and control of the
cytoskeleton. Some of these enzymes also act downstream
of receptor tyrosine kinases or G-protein-coupled
receptors.
Using two strategies to inhibit PI3K signaling in
embryos, the role of PI3Ks during early
Xenopus development has been analyzed. A class 1A PI3K
catalytic activity is required for the definition of trunk
mesoderm during the blastula stages, but is less important
for endoderm and prechordal plate mesoderm induction
or for organizer formation. It is required in the FGF
signaling pathway downstream of Ras and in parallel to
the extracellular signal-regulated kinase (ERK) MAP
kinases. In addition, ERKs and PI3Ks
can synergise to convert ectoderm into mesoderm. These
data provide the first evidence that class 1 PI3Ks are
required for a specific set of patterning events in vertebrate
embryos. Furthermore, they bring new insight into the
FGF signaling cascade in Xenopus (Carballada, 2001).
While PI3K has been shown to be required for signal
transduction in response to RTK ligands such as PDGF, insulin
or EGF, its role in FGF signaling is much less clear cut. On
the one hand, several molecules able to bind PI3K subunits,
such as dof,
act downstream of FGF or are found associated with the FGF
receptor. Also, treatment of cultured cell lines with basic FGF
can lead to a modest increase in PI3K activity. On the other hand, inhibition of
PI3K signaling seldom has a demonstrated direct effect on the
response to FGF and in the few cases where this appears to be
the case, the role of PI3K is limited to the reorganization of the
cytoskeleton or the regulation of exocytosis. In no
case has the direct activation by FGF of a target gene been shown to be PI3K dependent. In contrast to the controversial role of PI3K in FGF signaling, activation of the MAP kinase pathway plays a crucial role in
FGF signaling. On the basis of the overexpression of activated
MAP kinase, it has been suggested that the Ras-dependent activation of this kinase is sufficient to account for the FGF-mediated induction of mesoderm induction and for the direct activation of Xbra (Carballada, 2001).
Using two different strategies to interfere with PI3K
signaling, this study provides the first demonstration that PI3K
signaling is crucial for the direct activation by FGF of Xbra.
PI3K signaling is not involved in the activation of ERK by FGF
but rather acts in parallel to the MAP kinase pathway. In
contrast to what has been previously proposed, these results thus
indicate that, during mesoderm induction, the FGF signaling
pathway splits upstream of ERK into at least two cooperating
branches. Several questions remain to be addressed. (1) The
weak mesoderm induction obtained when both the ERK
and PI3K pathways are activated suggests the existence of
additional parallel effector pathways downstream of Ras.
Several effector pathways, including Ral, Rac/Rho and
phospholipase D, have been shown to act downstream of Ras
and probably in parallel to ERK and PI3K.
It will be important to test the role of these pathways in Xenopus
mesoderm formation. It will also be important to position PI3K
with respect to laloo, a recently described src-family tyrosine
kinase acting in the FGF pathway. (2) PI3K is required for FGF
signaling, whether this PI3K activity is modulated by FGF signaling has not been addressed. This could be the case, since in other systems FGF can stimulate, albeit weakly, PI3K activity. In addition, p85 is associated to the FGF receptor in Xenopus embryos during gastrulation. (3) The components acting downstream of PI3K in mesoderm induction must be identified. Several downstream effectors of PI3K have been
characterized in cultured cells including GSK3, PKB/Akt,
p70 S6k and the GTPases Rac and Rho. The results presented here do not support a role
for GSK3 downstream of PI3K in early embryos, since expression
of Siamois, a direct target of the beta-catenin/GSK3 pathway, is not affected by
treatment with LY294002. It will be interesting to test a
potential role for PKB/Akt and Rac/Rho in mesoderm induction
downstream of PI3K. The availability of constitutively active or
dominant negative forms of proteins acting in the Ras and PI3K
pathways in other systems, coupled with the convenience of the
Xenopus system, will help shed light on these issues (Carballada, 2001).
Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for
signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine renders this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restores its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3 (Hart, 2001).
This study examines the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibit the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85alpha oligonucleotide or LY294002, a selective inhibitor of PI3-kinase, independently inhibit the formation of erythropoietin-dependent colonies. In K562 cells, Src associates with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibits the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin-induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3-kinase in F-36P human erythroleukemia cells. The coexpression experiments and in vitro kinase assay further demonstrate that Src directly tyrosine-phosphorylates erythropoietin receptor, and associates with PI3-kinase. In vitro binding experiments have proven that glutathione S-transferase-p85alpha N- or C-terminal SH2 domains independently bind to erythropoietin receptor, which is tyrosine-phosphorylated by Src. Taken together, Src transduces the erythropoietin-induced erythroid differentiation signals by regulating PI3-kinase activity (Kubota, 2001).
Receptor tyrosine kinases (RTKs) play distinct roles in multiple biological
systems. Many RTKs transmit similar signals, raising questions about how
specificity is achieved. One potential mechanism for RTK specificity is control
of the magnitude and kinetics of activation of downstream pathways. The protein tyrosine phosphatase Shp2 regulates the strength and
duration of phosphatidylinositol 3'-kinase (PI3K) activation in the epidermal
growth factor (EGF) receptor signaling pathway. Shp2 mutant fibroblasts exhibit
increased association of the p85 subunit of PI3K with the scaffolding adapter
Gab1, compared to that for wild-type (WT) fibroblasts or Shp2 mutant cells
reconstituted with WT Shp2. Far-Western analysis suggests increased
phosphorylation of p85 binding sites on Gab1. Gab1-associated PI3K activity is
increased and PI3K-dependent downstream signals are enhanced in Shp2 mutant
cells following EGF stimulation. Analogous results are obtained in fibroblasts
inducibly expressing dominant-negative Shp2. These results suggest that, in
addition to its role as a positive component of the Ras-Erk pathway, Shp2 negatively regulates EGF-dependent PI3K activation by dephosphorylating Gab1 p85 binding sites, thereby terminating a previously proposed Gab1-PI3K positive feedback loop. Activation of PI3K-dependent pathways following stimulation by other growth factors is unaffected or decreased in Shp2 mutant cells. Thus, Shp2 regulates the kinetics and magnitude of RTK signaling in a receptor-specific manner (Zhang, 2002).
Chronic stimulation of norepinephrine (NE) neuromodulation by angiotensin II (Ang II) involves activation of the Ras-Raf-MAP kinase signal transduction pathway in Wistar Kyoto (WKY) rat brain neurons. This pathway is only partially responsible for this heightened action of Ang II in the spontaneously hypertensive rat (SHR) brain neurons. A MAP kinase-independent signaling pathway in the SHR neuron involves activation of PI3-kinase and protein kinase B (PKB/Akt). Ang II stimulates PI3-kinase activity in both WKY and SHR brain neurons and is accompanied by its translocation from the cytoplasmic to the nuclear compartment. Although the magnitude of stimulation by Ang II is comparable, the stimulation is more persistent in the SHR neuron, as compared to the WKY rat neuron. Inhibition of PI3-kinase has no significant effect in the WKY rat neuron. However, it causes a 40%-50% attenuation of the Ang II-induced increase in norepinephrine transporter (NET) and tyrosine hydroxylase (TH) mRNAs and [3H]-NE uptake in the SHR neuron. In contrast, inhibition of MAP kinase completely attenuates Ang II stimulation of NET and TH mRNA levels in the WKY rat neuron, whereas it causes only a 45% decrease in the SHR neuron. However, an additive attenuation is observed when both kinases of the SHR neurons are inhibited. Ang II also stimulates PKB/Akt activity in both WKY and SHR neurons. This stimulation is 30% higher and lasts longer in the SHR neuron, when compared with the WKY rat neuron. In conclusion, these observations demonstrate an exclusive involvement of PI3-kinase-PKB-dependent signaling pathway in a heightened NE neuromodulatory action of Ang II in the SHR neuron. Thus, this study offers an excellent potential for the development of new therapies for the treatment of centrally mediated hypertension (Yang, 1999).
Muscarinic acetylcholine receptor (mAChR), a member of the G-protein-coupled receptors (GPCRs) gene superfamily, has
been shown to mediate the effects of acetylcholine on differentiation and proliferation in the CNS. However, the mechanism or
mechanisms whereby mAChRs regulate cell proliferation remain poorly understood. In vitro bFGF-expanded
neural progenitor cells dissociated from rat cortical neuroepithelium express muscarinic acetylcholine receptor subtype mRNAs.
Stimulation of these mAChRs with carbachol, a muscarinic agonist, activates extracellular-regulated kinases
(Erk1/2) and phosphatidylinositol-3 kinase (PI-3K). This, in turn, stimulates DNA synthesis in neural progenitor cells. MEK inhibitor PD98059 and PI-3K inhibitors
wortmannin and LY294002 inhibit a carbachol-induced increase in DNA synthesis. These findings indicate that the activation of both PI-3 kinase and MEK
signaling pathways via muscarinic receptors is involved in stimulating DNA synthesis in the neural progenitor cells during early neurogenesis (Bing-Sheng, 2001).
The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous
system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high
affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF,
and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using
MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of
filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this
report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined.
Activation of RET results in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation.
This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002,
blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis (Tang, 2002).
ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. This study shows that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. These results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis (Nie, 2007).
The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras.
This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the
catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 [Raf (1-149) and
Raf (51-131)] were used. Not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a
concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex,
shows that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more
physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the
sequestration of Ras.GTP by Raf. Consistent with these results, the GEF-stimulated exchange of Ha-Ras.GTP is also inhibited by another effector of Ras, the RBD
(amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha. The data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream
activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell (Giglione, 1998).
Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. However, in vivo
there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the
plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent
activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the
Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimics the spacing of
Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The
mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized (Yan, 1998).
Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation. p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338. The origin of the signal for Pak-mediated Raf-1 activation has been investigated by examining the roles of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acts synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding are also readily activated by RasV12. This indirect activation of Raf-1 by Ras is blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI
3-kinase. In addition, p85delta, a mutant of the PI 3-kinase regulatory subunit, inhibits the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also block both activation and Ser338 phosphorylation of Raf-1 induced by
epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1 (Sun, 2000).
Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. This study generated mice with mutations in the Pi3kca gene, encoding the catalytic p110α isoform, that block p110α interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110α is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation (Gupta, 2007).
The creation of mice lacking the ability of their p110α PI 3-kinase catalytic subunit to interact with activated Ras provides an opportunity to definitively address the significance of this interaction in growth factor signaling both in vivo and in vitro. In cultured mouse embryo fibroblasts, loss of p110α binding to Ras strongly reduces PI 3-kinase activation by EGF and FGF-2, but not by PDGF. This differential requirement for Ras may reflect the fact that the activated receptor for PDGF directly binds the p85 regulatory subunit of PI 3-kinase at the plasma membrane, whereas the others do not. EGF receptor is thought to direct PI 3-kinase activation more indirectly, either via Gab1 and Grb2 or via ErbB3. In the case of FGF-2, its receptor phosphorylates the docking protein FRS2, which in turn binds Grb2 and Gab1. Where PI 3-kinase is recruited to receptor complexes indirectly, it is possible that smaller numbers of p110 molecules are activated, perhaps making a costimulatory role for Ras binding more essential. However, other considerations may also be involved, as insulin signaling to PI 3-kinase, which involves IRS family adaptors, does not appear to be majorly dependent on Ras interaction with p110α. Another possible explanation might be the extent to which particular growth factor receptors use different isoforms of p110. Deletion of p110α in mouse embryo fibroblasts results in more complete inhibition of EGF than PDGF signaling to Akt. It has been suggested that this might reflect an ability of PDGF, but not EGF, receptor to regulate p110β, which is also expressed in these cells (Gupta, 2007).
Mice have been made where a similar set of mutations has been introduced into a different isoform of PI 3-kinase, p110γ, whose expression is largely restricted to hematopoietic cells. This resulted in loss of accumulation of PIP3 in neutrophils in response to chemoattractants. In addition, flies with a similar mutation introduced into Dp110 show greatly reduced egg-laying ability and are small in size (Orme, 2006). This suggests that blocking Ras interaction with p110 can attenuate PI 3-kinase regulation in other systems as well (Gupta, 2007).
Interaction with Ras has been shown to allosterically activate PI 3-kinase via a change in the structure of the catalytic pocket, in a manner that is synergistic with binding of p85 to pYXXM peptides. By itself, the interaction of Ras with p110 is insufficient to drive membrane translocation of PI 3-kinase, a necessity for its activation, suggesting that the ability of oncogenic mutant Ras by itself to drive PI 3-kinase activation may be dependent on low-level signaling input from receptor tyrosine kinases, especially EGF receptor family members that respond to autocrine ligands produced in response to activation of the Raf/ERK branch of Ras downstream pathways (Gupta, 2007).
The phenotype of mice homozygous for the p110α RBD mutation suggests that in vivo there is also a requirement for the Ras/PI 3-kinase interaction for some growth factors to signal correctly. Most obviously, there is a partial failure and delay of development of the lymphatic system, resulting in the accumulation of chylous ascites in newborn pups. This developmental phenotype is similar to that of VEGF-C+/− mice and also to some extent angiopoietin 2 knockout mice. The sprouting of the first lymphatic vessels from embryonic veins appears to be highly dependent on VEGF-C signaling, with homozygous deletion of VEGF-C resulting in embryonic edema from E12.5, complete lack of lymphatic vasculature, and death in utero. The similarity of the phenotype of the VEGF-C heterozygotes and p110α RBD mutant homozygotes could suggest that failure of Ras to engage p110α directly might result in vivo in a roughly 50% reduction in the ability of VEGF-C to signal to a critical downstream effector system, such as PI 3-kinase/Akt. It has been demonstrated that VEGF-C promotes survival and proliferation of lymphatic endothelial cells in vitro and induces Akt and ERK activation. VEGF-C signals through two receptor tyrosine kinases, VEGFR2 and VEGFR3, of which VEGFR3 is critical for its role in control of lymphatic development. VEGFR3 makes a good candidate for a receptor that might require Ras to signal to PI 3-kinase as, like EGFR and FGFRs, it lacks good p85-binding motifs and presumably must engage the pathway indirectly. However, it is also possible that signaling through other lymphangiogenic growth factors and their receptors, such as angiopoeitin 2 and Tie2, could also be defective in the p110α RBD mutant mice (Gupta, 2007).
LEOPARD (LS) and Noonan (NS) are overlapping syndromes associated with distinct mutations of SHP-2. Whereas NS mutations enhance SHP-2 catalytic activity, the activity of three representative LS mutants is undetectable when assayed using a standard protein tyrosine phosphatase (PTP) substrate. A different assay using a specific SHP-2 substrate confirms their decreased PTP activity, but also reveals a significant activity of the T468M mutant. In transfected cells stimulated with epidermal growth factor, the least active LS mutants promote Gab1/PI3K binding, validating the in vitro data. LS mutants thus display a reduced PTP activity both in vitro and in transfected cells (Hanna, 2006).
Genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK) stimulated Ras/Map kinase signaling, through the recruitment of csw/ptp-2/Shp2. Using sensitised assays in C. elegans for let-23/Egfr and daf-2/InsR (Insulin receptor-like) signaling, it has been shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K) mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional because the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma mediated signaling (Hopper, 2006).
The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. This study identified DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. The gene for DEPDC6 is found only in vertebrates, and encodes a protein with tandem N-terminal DEP (dishevelled, egl-10, pleckstrin) domains and a C-terminal PDZ (postsynaptic density 95, discs large, zonula occludens-1) domain. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, this study identified a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival (Peterson, 2009).
Loss-of-function data indicate that DEPTOR inhibits both the mTORC1 and mTORC2 pathways. However, by inhibiting mTORC1, DEPTOR overexpression relieves mTORC1-mediated inhibition of PI3K, causing an activation of PI3K and, paradoxically, of mTORC2-dependent outputs, like Akt (Peterson, 2009).
mTOR interacts with DEPTOR via its PDZ domain, and so far there is no information about the function of the tandem DEP domains the protein also contains. In other proteins, DEP domains mediate protein-protein interactions, but in numerous DEPTOR purifications additional DEPTOR-interacting proteins were not identified, besides the known components of mTORC1 and mTORC2. Therefore, based on current evidence, DEPTOR appears dedicated to mTOR regulation, and it is proposed that in vertebrates it is likely to be involved in regulating other outputs of the mTOR signaling network besides the growth and survival pathways examined in this study. The mTOR complexes and DEPTOR negatively regulate each other, suggesting the existence of a feedforward loop in which the loss of DEPTOR leads to an increase in mTOR activity, which then further reduces DEPTOR expression. This type of regulatory circuit should result in DEPTOR expression being tightly coupled to mTOR activity, and, interestingly, it was noted that DEPTOR mRNA levels strongly anticorrelate with cell size, a readout of mTORC1 activity (Peterson, 2009).
About 28% of human multiple myelomas (MMs) overexpress DEPTOR. These results are consistent with a published survey of 67 MM tumors and 43 MM cell lines, in which 21% were shown to possess copy number gains and associated expression increases of the genes within a 6 Mb region of chromosome 8q24 that contains DEPTOR. Furthermore, it appears that deregulated overexpression of c-MAF and MAFB is an additional, perhaps even more prevalent, mechanism for increasing DEPTOR expression in MMs. The related c-MAF and MAFB transcription factors are expressed (frequently as the result of chromosomal translocations) in a large fraction of MMs, but not in the plasma cells from which they are derived. Consistent with c-MAF playing a key role in promoting DEPTOR expression, a knockdown of c-MAF in a MM cell line having a c-MAF translocation decreases the expression of DEPTOR and mimics the effects of a DEPTOR knockdown on mTOR and PI3K signaling. The levels of the DEPTOR and c-MAF or MAFB mRNAs highly correlate with each other and, importantly, DEPTOR expression correlates with poor survival in patients with multiple myeloma (Peterson, 2009).
In many multiple myeloma cell lines, DEPTOR is massively overexpressed compared to the levels found in other cancer cell lines, such as HeLa cells. In these cells, the great overexpression of DEPTOR inhibits mTORC1 growth signaling and drives outputs dependent on PI3K. Interestingly, a reduction in DEPTOR expression to the lower levels seen in non-multiple myeloma cell lines causes cell death via apoptosis. This suggests that a pharmacologically induced reduction in DEPTOR expression or disruption of the DEPTOR-mTOR interaction could have therapeutic benefits for the treatment of multiple myeloma. There has been progress in developing small-molecule inhibitors of protein-protein interactions mediated by PDZ domains, so it is conceivable that blockers of the DEPTOR-mTOR interaction could be made (Peterson, 2009).
Although a number of other cancer cell lines have high levels of DEPTOR, as a class only multiple myelomas appear to consistently overexpress it. Besides activating PI3K/Akt signaling, DEPTOR overexpression in MM cells may provide these cells with benefits that are not relevant in other cancer types or perhaps even detrimental. For example, the high demand that MM cells place on the protein synthesis machinery to produce large amounts of immunoglobulins, causes a significant ER stress, which renders these cells susceptible to apoptosis induction via agents that induce further ER stress, such as proteasome inhibitors. DEPTOR overexpression, by partial inhibition of protein synthesis through the suppression of mTORC1, may reduce the levels of ER stress below the threshold that triggers apoptosis. In contrast, in other cancer cells in which ER stress is not a significant factor, DEPTOR overexpression may be selected against because reduced rates of protein synthesis may not be tolerated. That mTORC1-stimulated protein synthesis leads to ER stress is already appreciated as TSC1 or TSC2 null cells have increased sensitivity to ER stress-induced death (Peterson, 2009).
It is curious that DEPTOR is overexpressed mostly in MMs characterized by chromosomal translocations instead of those that are hyperdiploid because of aneuploidy. Elevated DEPTOR expression might be tolerated better in the nonhyperdiploid MMs because aneuploidy itself increases sensitivity to conditions, like mTORC1 inhibition, that interfere with protein synthesis. Moreover, the state of high mTORC2 and low mTORC1 signaling that this work indicates that some MM cells prefer cannot be achieved by mutations that activate PI3K signaling, perhaps explaining why multiple myelomas exhibit low rates of PTEN-inactivating or PI3K-activating mutations (Peterson, 2009).
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