Xuejun Tian

Distinct Roles for Ras-Guanine Nucleotide-Releasing Factor 1 (Ras-GRF1) and Ras-GRF2 in the Induction of Long-Term Potentiation and Long-Term Depression

NMDA-type glutamate receptors (NMDARs) contribute to many forms of long-term potentiation (LTP) and long-term depression (LTD). NMDARs are heteromers containing calcium-permeating neuronal receptor 1 (NR1) subunits and a variety of NR2 subunits. Evidence suggests that, in the CA1 region of the hippocampus, NR2A-containing NMDARs promote LTP whereas NR2B-containing receptors promote LTD. However, the calcium sensors that distinguish between these signals to promote the appropriate form of synaptic plasticity are not known. Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) and Ras-GRF2 are highly similar calcium-stimulated exchange factors that activate Ras and Rac GTPases. Here, using a set of Ras-GRF knock-out mice, we show that Ras-GRF2 contributes predominantly to the induction of NMDAR-dependent LTP, whereas Ras-GRF1 contributes predominantly to the induction of NMDAR-dependent LTD in the CA1 region of the hippocampus of postpubescent mice (postnatal days 25–36). In contrast, neither Ras-GRF protein influences synaptic plasticity in prepubescent mice (postnatal days 14–18). Ras-GRF2 mediates signaling from (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl-phosphonic acid-sensitive (NVP-AAM077-sensitive) (NR2A-containing) NMDARs to the Ras effector extracellular signal-related protein kinase 1/2 (Erk1/2) mitogen-activated protein (MAP) kinase, a promoter of NMDAR-induced LTP at this site. In contrast, Ras-GRF1 mediates signaling from ifenprodil-sensitive (NR2B-containing) NMDARs to the Rac effector p38 MAP kinase, a promoter of LTD. These findings show that, despite their similar functional domain organization, Ras-GRF1 and Ras-GRF2 mediate opposing forms of synaptic plasticity by coupling different classes of NMDARs to distinct MAP kinase pathways. Moreover, the postnatal appearance of Ras-GRF-dependent LTP and LTD coincides with the emergence of hippocampal-dependent behavior, implying that Ras-GRF proteins contribute to forms of synaptic plasticity that are required specifically for mature hippocampal function.

Developmentally regulated role for Ras‐GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB

p140 Ras‐GRF1 and p130 Ras‐GRF2 constitute a family of calcium/calmodulin‐regulated guanine–nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras‐GRF1 and p130 Ras‐GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras‐GRFs and the known neuroprotective effect of CREB activity, ischemia‐induced CREB activation is reduced in the brains of adult Ras‐GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras‐GRF exchange factors, implying that Ras‐GRFs endow NMDARs with functions unique to mature neurons.

p130Cas Regulates the Activity of AND-34, a Novel Ral, Rap1, and R-Ras Guanine Nucleotide Exchange Factor

We previously identified a novel murine protein, AND-34, with a carboxyl-terminal domain homologous to Ras family guanine nucleotide exchange factors (GEFs), which bound to the focal adhesion docking protein p130Cas. Work by others has implicated both the human homologue of AND-34, BCAR3, and human p130Cas, BCAR1, in the resistance of breast cancer cells to the anti-estrogen tamoxifen. Here we report that AND-34 displays GEF activity on RalA, Rap1A, and R-Ras but not Ha-Ras GTPases in cells. In contrast to several other Ral-GEFs, the Ral GEF activity of AND-34 is not augmented by constitutively active Ha-RasVal-12, consistent with the absence of a detectable Ras-binding domain. Efficient binding to AND-34 required both the Src-binding domain and a flanking carboxyl-terminal region of p130Cas. The p130Cas-binding site mapped to a carboxyl-terminal sequence within the AND-34 GEF domain. Overexpression of p130Cas, but not an AND-34-binding mutant of p130Cas, inhibited the Ral GEF activity of co-transfected AND-34. This work identifies a new potential function for p130Cas and a new regulatory pathway involved in the control of Ral, Rap, and R-Ras GTPases that may participate in the progression of breast cancer cells to tamoxifen resistance.

PDK1 mediates growth factor‐induced Ral‐GEF activation by a kinase‐independent mechanism

Ras proteins transduce extracellular signals to intracellular signaling pathways by binding to and promoting the activation of at least three classes of downstream signaling molecules: Raf kinases, phosphoinositide‐3‐kinase (PI3‐K) and Ral guanine nucleotide exchange factors (Ral‐GEFs). Previous work has demonstrated that epidermal growth factor (EGF) activates Ral‐GEFs, at least in part, by a Ras‐mediated redistribution of the GEFs to their target, Ral‐GTPases, in the plasma membrane. Here we show that Ral‐GEF stimulation by EGF involves an additional mechanism, PI3‐K‐dependent kinase 1 (PDK1)‐induced enhancement of Ral‐GEF catalytic activity. Remarkably, this PDK1 function is not dependent upon its kinase activity. Instead, the non‐catalytic N‐terminus of PDK1 mediates the formation of an EGF‐induced complex with the N‐terminus of the Ral‐GEF, Ral‐GDS, thereby relieving its auto‐inhibitory effect on the catalytic domain of Ral‐GDS. These results elucidate a novel function for PDK1 and demonstrate that two Ras effector pathways cooperate to promote Ral‐GTPase activation.

Regulation of Ras Signaling Specificity by Protein Kinase C

ABSTRACT Ras proteins have the capacity to bind to and activate at least three families of downstream target proteins: Raf kinases, phosphatidylinositol 3 (PI 3)-kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). We have previously shown that the Ras/Ral-GEF and Ras/Raf pathways oppose each other upon nerve growth factor stimulation, with the former promoting proliferation and the latter promoting cell cycle arrest. Moreover, the pathways are not activated equally. While the Ras/Raf/Erk signaling pathway is induced for hours, the Ras/Ral-GEF/Ral signaling pathway is induced for only minutes. Here we show that this preferential down-regulation of Ral signaling is mediated, at least in part, by protein kinase C (PKC). In particular, we show that PKC activation by phorbol ester treatment of cells blocks growth factor-induced Ral activation while it enhances Erk activation. Moreover, suppression of growth factor-induced PKC activation enhances and prolongs Ral activation. PKC does not influence the basal activity of the Ral-GEF designated Ral-GDS but suppresses its activation by Ras. Interestingly, Ras binding to the C-terminal Ras binding domain of Ral-GDS is not affected by PKC activity. Instead, suppression of Ral-GDS activation occurs through the region N terminal to the catalytic domain, which becomes phosphorylated in response to phorbol ester treatment of cells. These findings identify a role for PKC in determining the specificity of Ras signaling by its ability to differentially modulate Ras effector protein activation.

Age-dependent participation of Ras-GRF proteins in coupling calcium-permeable AMPA glutamate receptors to Ras/Erk signaling in cortical neurons.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (AMPARs) are ligand-gated sodium channels. Through their ability to mediate the majority of rapid excitatory transmission in the central nervous system, these neurotransmitter receptors have been shown to influence synaptic plasticity. Some of these receptors are also calcium-permeable (CP), and they also have been implicated in regulating synaptic plasticity, particularly in interneurons where their concentration is highest. However, the biochemical pathways emanating from CP-AMPARs that mediate these effects have not been well characterized. In this paper, we show that CP-AMPARs are the predominant AMPAR class responsible for activating the Ras/Erk kinase signaling cascade and the cAMP-response element-binding protein (CREB) transcription factor in the cortex of mature mice. Activation of Ras and Erk, but not CREB, occurs through the calcium/calmodulin regulated Ras-GRF1 and Ras-GRF2 exchange factors, which form AMPA-induced complexes with CP-AMPARs but not calcium-impermeable (CI) AMPARs in vivo. Furthermore, we show that CP-AMPARs are also the major AMPAR type to activate Ras/Erk signaling in pubescent mice; however, at this developmental stage Ras-GRF (guanine nucleotide-releasing factor) proteins are not involved. Finally, in neonatal animals CI-AMPARs, but not CP-AMPARs, are the predominant AMPAR type that activates Ras-Erk signaling and CREB in cortical neurons. This occurs indirectly through activation of L-type voltage-dependent calcium channels, an event that is also Ras-GRF-independent. Thus, Ras/Erk signaling and CREB activity induced by AMPARs occur through age-dependent mechanisms that likely make unique developmentally dependent contributions to synaptic function.

Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

Background: The ability to distinguish similar experiences is a critical aspect of memory. Results: Contextual discrimination involves LTP promoted by calcium-permeable AMPA-type glutamate receptors, RAS-GRF1 and p38 MAP kinase. Conclusion: A newly discovered, LTP-supporting, signaling pathway contributes to a key component of memory formation. Significance: Defects in this signaling pathway may contribute to cognitive loss associated with neurological disorders. RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.