Ras oncogene at 85D
Signaling proteins from the same family can have markedly different roles in a given cellular context. Expression of one hundred constitutively active human small GTPases is found to induce cell morphologies that fall into nine distinct classes. An algorithm is developed for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues are mostly outside known interaction surfaces and are structurally far apart from one another. This study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants (Heo, 2003).
Fifty six of the expressed small GTPase constructs triggered no significant morphology changes, while 44 others induced marked morphology changes. The induced morphologies were clearly distinguishable from one another and fell into only nine distinct classes. The Rho family members Rho6, Rho7, RhoE, and ARHE induced a marked cell rounding. Cells transfected with CDC42, CDC42h, TC10, and TCL constructs showed extensions of thin processes that have been termed filopodia, while cells transfected with Rac1, Rac2, Rac3, and RhoG constructs extended lamellipodia that consisted of mostly circular membrane sheets. Transfection of RhoA, RhoB, and RhoC constructs induced polymerized actin bundles or stress fibers that reached across the cell. Only RhoD and RhoH did not show a significant morphology change (Heo, 2003).
Arf family small GTPases induced two types of morphologies. Several members of the Arl family induced a shrunken morphology, while Arf6 had one of the most distinct morphologies with multiple characteristics that include broader cell arms, local membrane spreading, filopodia extensions as well as actin polymerization throughout the cell body and along the cell periphery. Within the shrunken morphology class, Arl 1, Arl 2, and Arl 3 could be considered as a subclass with less pronounced shrinkage and occasional induction of short filopodia type processes that have been termed microspikes in other studies (Heo, 2003).
Cells transfected with Ras family small GTPases also show two distinguishable morphology classes. The oncogenic H-, K-, and N-Ras induce a marked polarized morphology with membrane ruffles and strong actin staining at a polar end of the cells, while cells transfected with most of the remaining members show cell spreading combined with hairlike filopodia formation with pronounced polymerized actin boutons at their ends. The spreading of these cells has a resemblance to eyelashes and looks markedly different from the morphology of lamellipodia induced by Rac or RhoG or the polarized morphology induced by Ras (Heo, 2003).
Finally, several of the Rab family members also have a strong effect on cell morphology. Rab4B, Rab13, Rab22A, Rab23, and Rab35 induce a local spread morphology characterized by local lamellipodia extensions and occasional filopodia induction. Rab8 and Rab8B have the most dramatic effect on cell morphology of all constructs tested and, like Arf6, fall into the multiple morphology class characterized by large branched structures with local lamellipodia and filopodia (Heo, 2003).
In conclusion, this study shows that the structural fold of Ras superfamily small GTPases can induce nine different morphology classes. Furthermore, the residues have been discovered that define the filopodia, lamellipodia, polar, and eyelash morphologies and it was unexpectedly found that the locations of the switch-of-function sites are mostly outside the known effector interaction surfaces and are far apart from each other. These engineered small GTPases with a changed functional selectivity will be useful as tools in pull-down assays to identify the function-specific binding partners as perturbation constructs to investigate crosstalk between signaling processes and for testing whether particular cell functions are physiologically relevant by creating mutant model organisms. Finally, this study introduced an algorithm and a genome-based experimental classification strategy that can be employed to classify the functional space of protein families and to understand the structural basis of functional specificity (Heo, 2003).
Many cellular signaling proteins contain SH3 (Src homology 3) domains
that mediate protein interactions via specific proline-containing peptides.
Unlike SH2 domains, whose interactions with tyrosine-containing
peptides are promoted by phosphorylation of the SH2 binding site, the
regulatory mechanism for SH3 interactions is unclear. (Note: What is Src? Src is an oncogene whose protein interaction domains, SH2 and SH3, are the prototypes for similar domains in other proteins- see Drosophila Src64). p120 RasGAP (GTPase-activating protein: see Drosophila GTPase-activating protein 1), which contains an SH3 domain flanked by two SH2 domains, forms an abundant SH2-mediated complex with p190
RhoGAP in cells expressing activated tyrosine kinases. Two closely linked tyrosine-containing peptides have been identified in p190 that
bind simultaneously to the RasGAP SH2 domains upon p190
phosphorylation. This interaction is expected to bring the two SH2
domains into close proximity. Consequently, RasGAP undergoes a
conformational change that results in a 100-fold increase in the
accessibility of the target binding surface of its SH3 domain. These
results indicate that the tandem arrangement of SH2 and SH3 domains
found in a variety of cellular signaling proteins can provide a
conformational mechanism for regulating SH3-dependent interactions
through tyrosine phosphorylation. In addition, it appears that the role of
p190 in the RasGAP signaling complex is to promote additional protein
interactions with RasGAP via its SH3 domain (Hu, 1997).
The v-Src oncoprotein perturbs the dynamic regulation of the cellular cytoskeletal and adhesion network by a mechanism that is poorly understood. The effects of a temperature-dependent v-Src protein were examined on the regulation of p190 RhoGAP, a GTPase activating protein (GAP) that has been implicated in disruption of the organized actin cytoskeleton. Also addressed was the dependence of v-Src-induced stress fiber loss on inhibition of Rho activity. Activation of v-Src induces association of tyrosine phosphorylated p190 with p120(RasGAP) and stimulation of p120(RasGAP)-associated RhoGAP activity, although p120(RasGAP) itself is not a target for phosphorylation by v-Src in chicken embryo cells. These events require the catalytic activity of v-Src and are linked to loss of actin stress fibers during morphological transformation and not mitogenic signaling. Furthermore, these effects are rapidly reversible since switching off v-Src leads to dissociation of the p190/p120(RasGAP) complex, inactivation of p120(RasGAP)-associated RhoGAP activity and re-induction of actin stress fibers. In addition, transient transfection of Val14-RhoA, a constitutively active Rho protein that is insensitive to RhoGAPs, suppresses v-Src-induced stress fiber loss and cell transformation. Thus, an activated Src kinase requires the inactivation of Rho-mediated actin stress fiber assembly to induce its effects on actin disorganization. This work supports p190 as a strong candidate effector of v-Src-induced cytoskeletal disruption, most likely mediated by antagonism of the cellular function of Rho (Fincham, 1999).
Using the yeast two-hybrid system, developmentally
regulated Dictyostelium genes have been identified whose encoded proteins interact with Ras.GTP
but not Ras.GDP. One of
these genes encodes a homolog of Ras GAP (DdRasGAP1). Cells carrying a DdRasGAP1
gene disruption (ddrasgap1 null cells) have multiple, very distinct growth and
developmental defects. (1) Vegetative ddrasgap1 null cells are very large
and highly multinucleate cells when grown in suspension, indicating a severe
defect in cytokinesis.
The multinucleate phenotype, combined with results indicating that
constitutive expression of activated Ras does not yield highly multinucleate
cells, along with data on Ras null mutants, suggest that Ras may need to cycle
between GTP- and GDP-bound states for proper cytokinesis. (2) ddrasgap1 null cells also have multiple developmental
phenotypes that indicate an essential role of DdRasGAP1 in controlling cell
patterning. Multicellular development is normal through the mid-slug stage,
after which morphological differentiation is very abnormal and no culminant is
formed: no stalk cells and very few spores are detected. Proper DdRasGAP1 function and possibly
normal Ras activity are necessary to maintain spatial organization and for
induction of prestalk to stalk and prespore to spore cell differentiation. The
inability of ddrasgap1 null cells to initiate terminal differentiation and form
stalk cells is consistent with a model in which Ras functions as a mediator of
inhibitory signals in cell-type differentiation at this stage. (3) DdRasGAP1
and cAMP dependent protein kinase (PKA) interact to control spatial
organization within the organism. Overexpression of the PKA catalytic
subunit in ddrasgap1 cells yields terminal structures that are multiply
branched but lack spores. These growth and development defects suggest that RasGAP and PKA may mediate
common pathways that regulate apical tip differentiation and organizer
function, which in turn control spatial organization during multicellular
development. They also suggest that DdRasGAP1 either lies downstream from
PKA in the prespore to spore pathway or in a parallel pathway that is also
essential for spore differentiation (S. Lee, 1997).
During induction of the Caenorhabditis elegans hermaphrodite vulva, a signal from the anchor cell
activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase
signaling pathway in the vulval precursor cells. Two mechanisms have been characterized that limit the
extent of vulval induction. (1) gap-1 may directly inhibit the LET-60 Ras signaling
pathway. The gap-1 gene was identified in a genetic screen for inhibitors of vulval induction. gap-1 is
predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the
signaling activity of LET-60 Ras. A loss-of-function mutation in gap-1 suppresses the vulvaless
phenotype of mutations in the let-60 ras signaling pathway, but a gap-1 single mutant does not exhibit
excess vulval induction. (2) It was found that let-23 EGFR prevents vulval induction in a
cell-nonautonomous manner, in addition to its cell-autonomous role in activating the let-60 ras/mpk-1
signaling pathway. Using genetic mosaic analysis, it has been shown that let-23 activity in the vulval precursor
cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the
anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells
might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive
signal to distal vulval precursor cells (Hajnal, 1997).
The gene encoding p120-rasGAP, a negative regulator of Ras, has been disrupted in
mice. This Gap mutation affects the ability of endothelial cells to organize into a highly
vascularized network and results in extensive neuronal cell death. Mutations in the
Gap and Nf1 genes have a synergistic effect, such that embryos homozygous for
mutations in both genes show an exacerbated Gap phenotype. Thus rasGAP and
neurofibromin act together to regulate Ras activity during embryonic development (Henkemeyer, 1995).
The three-dimensional structure of the complex between human H-Ras bound to guanosine
diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human
GTPase-activating protein p120(GAP) in the presence of aluminum fluoride was solved at
a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature
of the communication between the two molecules, which explains the sensitivity of the interaction
toward both salts and lipids. An arginine side chain (arginine-789) of p120 was supplied into the
active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is
stabilized by, thus allowing glutamine-61 of Ras (a mutation that activates the oncogenic
potential) to participate in catalysis. The structural arrangement in the active site is consistent with a
mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of
Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der
Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras; even its mutation to
alanine would disturb the arrangements of residues in the transition state (Scheffzek, 1997).
Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton
following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an
SH3 domain and two SH2 domains, and there is evidence that they harbor a signaling function. A monoclonal antibody to the SH3 domain of Ras-GAP blocks Ras signaling in Xenopus oocytes. Microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by
growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho,
suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks
lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner,
strongly suggesting an effector function, in addition to its role as a Ras downregulator. These results support the idea that
Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism that
probably does not involve p190 Rho-GAP (Leblanc, 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 has led to the suggestion that GAP1(m) might function as a
PIP3 receptor in vivo. Using rat pheochromocytoma PC12 cells transiently transfected with a plasmid expressing
a chimera of green fluorescent protein fused to GAP1(m) [GFP-GAP1(m)], it has been shown 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, and it is recruited to the plasma membrane, but does not appear to be activated, following
agonist stimulation of PI 3-kinase (Lockyer, 1999).
The role of Ras GTPase-activating protein (GAP) has been investigated in
NGF-induced neuronal differentiation by overexpressing both wild-type and
membrane-targeted GAP in PC12 cells. Extension of neurites in response to NGF
is completely blocked in cells expressing the highest level of membrane-targeted
GAP and significantly inhibited in cells expressing either wild-type GAP or lower
levels of membrane-targeted GAP. Overexpression of membrane-targeted GAP
similarly inhibits induction of differentiation by src, but not by ras or raf oncogenes,
indicating that GAP inhibits differentiation of PC12 cells by downregulating Ras
function. GAP overexpression also inhibits stimulation of mitogen-activated protein
(MAP) kinase and induction of immediate-early genes in response to NGF. In cells
expressing wild-type GAP or lower levels of membrane-targeted GAP, the initial
activation of MAP kinase and immediate-early gene expression are only partially
inhibited. However, GAP expression in these cells results in substantial inhibition of
sustained MAP kinase activity following NGF treatment, consistent with the inhibition
of neurite extension in these cell lines. These results indicate that GAP acts as a
negative regulation, rather than an effector, of Ras signaling in PC12 cells (Yao, 1995).
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are signal transducers that relay messages from many receptors on the cell surface to
modulate various cellular processes. The direct downstream effectors of G proteins consist of the signaling molecules that are activated by their physical
interactions with a G alpha or Gbetagamma subunit. Effectors that interact directly with G alpha12 G proteins have yet to be identified. G
alpha12 binds directly to, and stimulates the activity of, Bruton's tyrosine kinase (Btk) and a Ras GTPase-activating protein, Gap1m, in vitro and in vivo. G
alpha12 interacts with a conserved domain, composed of the pleckstrin-homology domain and the adjacent Btk motif that is present in both Btk and Gap1m.
These results are the first to identify direct effectors for G alpha12 and to show that there is a direct link between heterotrimeric and
monomeric G proteins (Jiang, 1998).
A 62 kDa protein is highly phosphorylated in many cells containing activated tyrosine
kinases. This protein, characterized mainly by its avid association with rasGAP, has
proved difficult to identify. In this study, anti-phosphotyrosine antibody was used to purify p62. Based on its peptide
sequence, molecular cloning reveals a cDNA encoding a novel protein, p62dok, with
little homology to other proteins but with a prominent set of tyrosines and nearby sequences
suggestive of SH2 binding sites. v-Abl (See Drosophila Abl Oncogene) tyrosine kinase binds and strongly
phosphorylates p62dok, which then binds rasGAP. 2C4A, a monoclonal antibody to the
rasGAP-associated p62, reacts with p62dok. Thus, p62dok appears to be the
long-sought major substrate of many tyrosine kinases (Yamanashi, 1997).
Dok, a 62-kDa Ras GTPase-activating protein, in other words, a rasGAP-associated phosphotyrosyl protein, is thought to act as a multiple docking protein
downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induces marked tyrosine phosphorylation of
Dok. This adhesion-dependent phosphorylation of Dok is mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced
tyrosine phosphorylation of Dok requires a Src family kinase. A mutant Dok (DokDeltaPH) that lacks its pleckstrin homology domain fails to
undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH does not localize to
subcellular membrane components. Insulin promotes the association of tyrosine-phosphorylated Dok with the adapter protein NCK (Drosophila homolog: (Dreadlocks) and rasGAP. In
contrast, a mutant Dok (DokY361F), in which Tyr361 is replaced by phenylalanine, fails to bind NCK but partially retains the ability to bind
rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhances the cell migratory response
to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially
links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility (Noguchi, 1999).
The mechanism of v-src-induced morphological transformation is still obscure. LA29 rat
fibroblasts, which express a temperature-sensitive (ts) v-src mutant, were compared with D1025 rat fibroblasts,
transfected with a ts mutant of v-fps. Upon transformation, LA29 cells adopt an elongated shape
with reduced focal adhesions and loss of actin stress fibers. In contrast, activation of v-fps in D1025
cells has little effect on morphology. In both cells, paxillin is strongly tyrosine phosphorylated upon
activation of the kinases. This indicates that paxillin phosphorylation is either not required, or not sufficient, for
the v-src-induced disruption of focal adhesions. v-src activates the
ras-MAP kinase (MAPK) pathway, as indicated by tyrosine phosphorylation of the rasGAP-associated
proteins p62 and p190 and MAPK phosphorylation. Since MAPK affects transcription, this suggests
that novel gene transcription is required. This notion was confirmed using actinomycin D and
cycloheximide, which do not impair activation of v-src kinase activity, but completely block
v-src-induced morphological changes, as demonstrated using image analysis. v-src-induced changes in cell shape occur before the reduction in number and size of
focal adhesions. It is concluded that v-src-induced transformation of rat fibroblasts depends on synthesis
of a protein, which induces rapid changes in cell shape that precede the loss of focal adhesions (Meijne, 1997).
Neurofibromin, a GTPase-activating protein, plays a critical role in the downregulation of Ras proteins in neurons
and Schwann cells. The ability of neurofibromin to interact with Ras is crucial for
its function, since mutations in NF1 that abolish this interaction fail to maintain function.
A yeast two-hybrid screen was carried out using a mutant of H-ras, H-rasD92K, defective
for interaction with the GTPase-activated protein-related domain (GRD) of NF1. Two
screens of a randomly mutagenized NF1-GRD library led to the identification of seven
novel NF1 mutants. Characterization of the NF1-GRD mutants reveals that one
class of mutants is allele specific for H-raSD92K. These mutants exhibit increased
affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein.
They do not interact with another H-ras mutant defective for interaction
with GTPase-activating proteins. Another class consists of high-affinity mutants
that exhibit dramatically increased affinity for both wild-type and mutant forms of
Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of
activated RAS2G19v in yeast cells. Five mutations cluster within a region
encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have
previously been identified in this region. Two mutations occur in a
region encompassing residues 1262 to 1276 (region I). Combining high-affinity
mutations from both regions results in even greater affinity for Ras. These results
demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction
and that single amino acid changes can affect NF1's affinity for Ras (Morcos, 1996).
Schwann cells are thought to be abnormal in type 1 neurofibromatosis (NF1) and to
contribute to the formation of benign and malignant tumors in this disease. To test the
role of the NF1 gene product neurofibromin as a Ras-GTPase activating protein in
Schwann cells, and to study the effect of the loss of neurofibromin on Schwann cell
proliferation, Schwann cells were isolated from mice with targeted disruption of NF1.
The properties of these neurofibromin deficient cells are strikingly similar to those of
v-ras expressing rat Schwann cells with normal levels of neurofibromin. The
similarities include: growth inhibition, noted as a decrease in both cell division in response
to glial growth factor 2 (GGF2) and in neuronal contact; morphological changes such
as the appearance of elaborated processes, and elevated levels of Ras-GTP.
Ras-GTP levels in the neurofibromin deficient Schwann cells are
consistently elevated in response to GGF2 treatment. In contrast to these results,
introduction of v-ras into a Schwannoma cell line (RN22) leads to cell transformation.
It is concluded that neurofibromin functions as a major regulator of Ras-GTP in
Schwann cells; however, mutation in NF1 by itself is unlikely to explain the
hyperplasia observed in Schwann cell tumors in NF1 disease (Kim, 1995).
The PSD-95/SAP90 family of proteins has recently been implicated in the organization of synaptic structure. A novel Ras-GTPase activating protein, SynGAP, has been isolated that interacts with the PDZ domains of PSD-95 and SAP102 in vitro and in vivo. SynGAP is selectively expressed in brain and is highly enriched at excitatory synapses, where it is present in a large macromolecular complex with PSD-95 and the NMDA receptor. SynGAP stimulates the GTPase activity of Ras, suggesting that it negatively regulates Ras activity at excitatory synapses. Ras signaling at the postsynaptic membrane may be involved in the modulation of excitatory synaptic transmission by NMDA receptors and neurotrophins. These results indicate that SynGAP may play an important role in the modulation of synaptic plasticity (Kim, 1998).
Synaptic NMDA-type glutamate receptors are anchored to the second of three PDZ (PSD-95/Discs large/ZO-1) domains in the postsynaptic
density (PSD) protein PSD-95. Citron, a protein target for the activated form of the small GTP-binding protein Rho,
preferentially binds the third PDZ domain of PSD-95. In GABAergic neurons from the hippocampus, citron forms a complex with PSD-95
and is concentrated at the postsynaptic side of glutamatergic synapses. Citron is expressed only at low levels in glutamatergic neurons in the
hippocampus and is not detectable at synapses onto these neurons. In contrast to citron, both p135 SynGAP (an abundant synaptic Ras
GTPase-activating protein that can bind to all three PDZ domains of PSD-95) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase
II) are concentrated postsynaptically at glutamatergic synapses on glutamatergic neurons.
SynGAP, a Ras GTPase activating protein, is nearly as abundant in the PSD fraction as
PSD-95 itself. SynGAP can be phosphorylated by Ca2+/calmodulin-dependent protein kinase
II (CaM kinase II) in the PSD fraction and its GAP activity is reduced after phosphorylation. Thus, SynGAP and CaM kinase II constitute a signal transduction complex associated
with the NMDA receptor. CaM kinase II is not expressed and p135 SynGAP
is expressed in less than half of hippocampal GABAergic neurons. Segregation of citron into inhibitory neurons does not occur in other brain
regions. For example, citron is expressed at high levels in most thalamic neurons, which are primarily glutamatergic and contain CaM kinase
II. In several other brain regions, citron is present in a subset of neurons that can be either GABAergic or glutamatergic and can sometimes
express CaM kinase II. Thus, in the hippocampus, signal transduction complexes associated with postsynaptic NMDA receptors are different
in glutamatergic and GABAergic neurons and are specialized in a way that is specific to the hippocampus (Zhang, 1999).
The results presented
here support the notion that differential expression of PSD-95-binding proteins in different neurons
helps to determine the composition of signal transduction complexes formed by association with
PSD-95 at glutamatergic PSDs. The resulting distinct compositions of these complexes will likely define
the nature of local biochemical signaling associated with activation of NMDA receptors.
The selective localization of citron suggests that, in hippocampus, PSDs of glutamatergic
synapses made onto inhibitory interneurons contain cytoskeletal regulatory machinery that is not present
at glutamatergic synapses made onto excitatory principal neurons. Furthermore, CaM kinase II is not
detectable in these same PSDs but is present in the postsynaptic complex of excitatory synapses made
onto glutamatergic neurons in the hippocampus. CaM
kinase II can phosphorylate and regulate the GluRA/1 subunit of AMPA-type glutamate receptors and the synaptic Ras GTPase-activating protein SynGAP and can phosphorylate the NR2A and NR2B subunits of the NMDA receptor. This regulation by CaM kinase II is absent from the postsynaptic side of
glutamatergic synapses on hippocampal inhibitory neurons. Thus, the modes of regulation of synaptic
structure (by citron) and of synaptic strength (by CaM kinase II or citron) at glutamatergic synapses
will differ dramatically between excitatory and inhibitory neurons.
High citron expression found only in GABAergic neurons appears to be a unique feature of the hippocampus.
In other brain regions, such as the thalamus and cerebral cortex, citron and CaM kinase II are often
found together in excitatory neurons. Thus, the composition of signal transduction
machinery at the postsynaptic membrane of glutamatergic synapses varies among neurons throughout
the brain in ways that cannot be classified simply. Furthermore, findings regarding the mechanisms of
signal transduction and plasticity at hippocampal synapses may not always generalize to synapses in
other areas of the brain (Zhang, 1999).
Ca2+ influx through n-methyl-d-aspartate- (NMDA-) type glutamate receptors plays a critical role in
synaptic plasticity in the brain. One of the proteins activated by the increase in Ca2+ is CaM kinase II
(CaMKII). A novel synaptic Ras-GTPase activating protein (p135 SynGAP) is described that is a
major component of the postsynaptic density, a complex of proteins associated with synaptic NMDA
receptors. The sequence contains four regions homologous to previously identified protein motifs.
Most notable is the RasGAP motif from positions 393 to 717. Its sequence is 30% identical
to p120 RasGAP and it contains the FLR PA P motif diagnostic for RasGAPs. The amino-terminal segment contains a putative PH domain, which may attach the protein to the membrane, and a region 31% identical to the C2 domain of p120 GAP, a motif that mediates binding to phospholipid and/or Ca2+ in synaptotagmin and
protein kinase C. Two of the putative splice variants end in QTRV,
conforming to the consensus sequence tS/TXV, which can bind to the second and third PDZ domains of
the scaffold protein PSD-95. A proline-rich region between positions 770 and
800 may form a binding site for SH3 domains. The message encoding p135 SynGAP is expressed at higher levels in brain than in other tissues. Furthermore, within neurons the protein is highly localized to synapses. Western blots of subcellular fractions from rat forebrain made with antibodies against p135 SynGAP reveal that it is enriched in isolated PSDs even after extraction with the relatively harsh detergent N-lauroyl sarcosinate. p135 SynGAP is almost exclusively localized at synapses in hippocampal neurons, where it binds to and closely colocalizes with the scaffold protein PSD-95 and colocalizes with NMDA receptors. The Ras-GTPase activating activity of p135 SynGAP is inhibited by phosphorylation by CaMKII located in the PSD protein complex. Inhibition of p135 SynGAP by CaMKII will stop inactivation of GTP-bound Ras and thus could result in activation of the mitogen-activated protein
(MAP) kinase pathway in hippocampal neurons upon activation of NMDA receptors (Chen, 1998).
Plexins serve as receptors for repulsive axonal guidance molecules semaphorins. The cytoplasmic domain of the semaphorin 4D (Sema4D) receptor, Plexin-B1 has two separated Ras GTPase-activating protein (GAP)-homologous domains, C1 and C2. The Rho family small GTPase Rnd1 associates with Plexin-B1, and the Plexin-B1-Rnd1 complex stimulates GTPase activity of R-Ras, inducing growth cone collapse in hippocampal neurons in response to Sema4D. However, the molecular mechanisms by which Plexin-B1 exhibits the GAP activity remain unclear. This study examines critical roles of Rnd1 and Sema4D in Plexin-B1-stimulated R-Ras GAP activity and neurite remodeling. The N-terminal region of the cytoplasmic domain of Plexin-B1 containing the C1 domain interacts with the C-terminal region containing the C2 domain, and Rnd1 disrupts this interaction. In contrast, Sema4D induces clustering of Rnd1-bound Plexin-B1, in parallel with inactivation of R-Ras in cells. Antibody clustering of the recombinant cytoplasmic domain of Plexin-B1 in the presence of Rnd1 triggers the R-Ras GAP activity. Deletion of the extracellular domain of Plexin-B1 causes ligand-independent clustering of the receptor, rendering the receptor constitutively active in the presence of Rnd1, and induces contraction of COS-7 cells and inhibition of neurite outgrowth in hippocampal neurons. These results indicate that Rnd1 opens the two R-Ras GAP domains of Plexin-B1, and Sema4D-induced receptor clustering stimulates R-Ras GAP activity and neurite remodeling in hippocampal neurons (Oinuma, 2004).
Plexins are receptors for the axonal guidance molecules known as semaphorins, and the semaphorin 4D (Sema4D) receptor plexin-B1 induces repulsive responses by functioning as an R-Ras GTPase-activating protein (GAP). This study characterized the downstream signalling of plexin-B1-mediated R-Ras GAP activity, inducing growth cone collapse. Sema4D suppresses R-Ras activity in hippocampal neurons, in parallel with dephosphorylation of Akt and activation of glycogen synthase kinase (GSK)-3beta. Ectopic expression of the constitutively active mutant of Akt or treatment with GSK-3 inhibitors suppressea the Sema4D-induced growth cone collapse. Constitutive activation of phosphatidylinositol-3-OH kinase (PI(3)K), an upstream kinase of Akt and GSK-3beta, also blocka the growth cone collapse. The R-Ras GAP activity is necessary for plexin-B1-induced dephosphorylation of Akt and activation of GSK-3beta and is also required for phosphorylation of a downstream kinase of GSK-3beta, collapsin response mediator protein-2. Plexin-A1 also induces dephosphorylation of Akt and GSK-3beta through its R-Ras GAP activity. It is concluded that plexin-B1 inactivates PI(3)K and dephosphorylates Akt and GSK-3beta through R-Ras GAP activity, inducing growth cone collapse (Ito, 2006).
Plexins are receptors for axonal guidance molecules semaphorins. The semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). This study examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. It is also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 alpha activity. Thus, it is proposed that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse (Oinuma, 2010).
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Ras85D:
Biological Overview
| Regulation
| Protein Interactions
| Effects of Mutation
| Ras as Oncogene
| References
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