RC3/neurogranin negatively regulates extracellular signal-regulated kinase pathway through its interaction with Ras.

RC3/neurogranin is a postsynaptic protein and plays pivotal roles in spatial learning and emotional anxiety as well as synaptic plasticity. The expression level of RC3 is dynamically changed during developmental stages, but the function of RC3 in brain development is not well understood yet. Neurotrophins interact with tropomyosin-related kinase receptors to activate Ras-extracellular signal-regulated kinase (ERK) pathway and can also induce neuronal differentiation. In this study, we demonstrate that RC3 inhibits Ras-ERK pathway by interaction with Ras and controls neurite outgrowth induced by neurotrophins. In PC12 cells, RC3 inhibits nerve growth factor (NGF)-induced activation of Ras and thereby ERK1/2 signaling cascade as well as neurite outgrowth induced by NGF. We found Ras is the target of the inhibitory function of RC3, because RC3 interacts with Ras and suppresses the elevated affinity of Ras to Ras-binding domain of Raf-1. Meanwhile, already activated Raf-1 by Ras activity is not affected by RC3. Furthermore, depletion of RC3 by RNA interference drastically enhances the stimulation of ERK1/2 and neurite outgrowth induced by brain-derived neurotrophic factor in hippocampal neurons. These findings suggest that RC3 is a novel natural inhibitor of Ras-ERK1/2 signaling axis, leading to negatively regulate neuronal differentiation induced by neurotrophins.

CIIA negatively regulates neuronal cell death induced by oxygen-glucose deprivation and reoxygenation.

Brain ischemia causes neuronal injury leading to stroke and other related brain diseases. However, the precise mechanism of the ischemia-induced neuronal death remains unclear yet. In this study, we showed that CIIA suppressed neuronal cell death induced by oxygen and glucose deprivation followed by reoxygenation (OGD/R), which mimics ischemia and reperfusion in vivo, in neuroblastoma cell lines as well as primary cortical neurons. Furthermore, CIIA inhibited the OGD/R-induced stimulation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream kinases including c-Jun amino-terminal kinase and p38 kinase, concomitantly blocking ASK1 homo-oligomerization and the binding between ASK1 and TRAF2. CIIA also repressed the OGD/R-induced activation of caspase-3 in neuronal cells. Taken together, our results suggest that CIIA attenuates neurotoxicity caused by OGD/R through inhibiting ASK1-dependent signaling events.

CIIA prevents SOD1(G93A)-induced cytotoxicity by blocking ASK1-mediated signaling.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease with higher selectivity in degeneration of motor neurons. However, the molecular mechanism by which the ALS-linked mutants of human superoxide dismutase 1 (SOD1) gene induce neurotoxicity remains obscure yet. Here, we show that depletion of CIIA expression by RNA interference (RNAi) promoted cytotoxicity caused by ALS-linked G93A mutant of the SOD1 gene. The RNAi-mediated knockdown of CIIA also enhanced the SOD1(G93A)-induced interaction between ASK1 and TRAF2 as well as ASK1 activity. Furthermore, endogenous silencing of CIIA by RNAi augmented the effects of SOD1(G93A) on reduction of mitochondria membrane potential (Δψm), release of cytochrome c into the cytoplasm, and caspase activation. Together, our results suggest that CIIA negatively modulates ASK1-mediated cytotoxic signaling processes in a SOD1(G93A)-expressing cellular model of ALS.

CIIA negatively regulates the Ras-Erk1/2 signaling pathway through inhibiting the Ras-specific GEF activity of SOS1.

Son of sevenless 1 (SOS1) is a Ras-specific guanine-nucleotide-exchange factor (GEF) that mediates intracellular signaling processes induced by receptor tyrosine kinases. In this study, we show that CIIA (also known as VPS28) physically associates with SOS1 and thereby inhibits the GEF activity of SOS1 on Ras, which prevents the epidermal growth factor (EGF)-induced activation of the Ras-Erk1/2 pathway. Furthermore, CIIA inhibited cyclin D1 expression, as well as DNA, synthesis in response to EGF. Intriguingly, CIIA failed to inhibit the Ras-specific GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L and E846K). Taken together, our results suggest that CIIA functions as a negative modulator of the SOS1-Ras signaling events initiated by peptide growth factors including EGF.

MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species-dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.

CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1.

Son of sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the guanosine triphosphatases Rac1 and Ras, which mediate signaling initiated by peptide growth factors. In this paper, we show that CIIA is a new binding partner of SOS1. CIIA promoted the SOS1-Rac1 interaction and inhibited the SOS1-Ras interaction. Furthermore, CIIA promoted the formation of an SOS1-EPS8 complex and SOS1-mediated Rac1 activation, whereas it inhibited SOS1-mediated activation of Ras. Transforming growth factor ß (TGF-ß) up-regulated the expression of CIIA and thereby promoted the association between CIIA and SOS1 in A549 human lung adenocarcinoma cells. Depletion of CIIA in these cells by ribonucleic acid interference inhibited the TGF-ß-induced interaction between SOS1 and EPS8, activation of Rac1, and cell migration. Together, these results suggest that CIIA mediates the TGF-ß-induced activation of SOS1-Rac1 signaling and cell migration in A549 cells. They further show that CIIA functions as a molecular switch for the GEF activity of SOS1, directing this activity toward Rac1.

Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling.

Microglia, the resident macrophages of the mammalian central nervous system, migrate to sites of tissue damage or infection and become activated. Although the persistent secretion of inflammatory mediators by the activated cells contributes to the pathogenesis of various neurological disorders, most activated microglia eventually undergo apoptosis through the process of activation-induced cell death (AICD). The molecular mechanism of AICD, however, has remained unclear. Here, we show that Daxx and mammalian Ste20-like kinase-1 (MST1) mediate apoptosis elicited by interferon-γ (IFN-γ) in microglia. IFN-γ upregulated the expression of Daxx, which in turn mediated the homodimerization, activation, and nuclear translocation of MST1 and apoptosis in microglial cells. Depletion of Daxx or MST1 by RNA interference also attenuated IFN-γ-induced cell death in primary rat microglia. Furthermore, the extent of IFN-γ-induced death of microglia in the brain of MST1-null mice was significantly reduced compared with that apparent in wild-type mice. Our results thus highlight new functions of Daxx and MST1 that they are the key mediators of microglial cell death initiated by the proinflammatory cytokine IFN-γ.

Negative regulation of SEK1 signaling by serum- and glucocorticoid-inducible protein kinase 1.

Serum- and glucocorticoid-inducible protein kinase 1 (SGK1) has been implicated in diverse cellular activities including the promotion of cell survival. The molecular mechanism of the role of SGK1 in protection against cellular stress has remained unclear, however. We have now shown that SGK1 inhibits the activation of SEK1 and thereby negatively regulates the JNK signaling pathway. SGK1 was found to physically associate with SEK1 in intact cells. Furthermore, activated SGK1 mediated the phosphorylation of SEK1 on serine 78, resulting in inhibition of the binding of SEK1 to JNK1, as well as to MEKK1. Replacement of serine 78 of SEK1 with alanine abolished SGK1-mediated SEK1 inhibition. Oxidative stress upregulated SGK1 expression, and depletion of SGK1 by RNA interference potentiated the activation of SEK1 induced by oxidative stress in Rat2 fibroblasts. Moreover, such SGK1 depletion prevented the dexamethasone-induced increase in SGK1 expression, as well as the inhibitory effects of dexamethasone on paclitaxel-induced SEK1-JNK signaling and apoptosis in MDA-MB-231 breast cancer cells. Together, our results suggest that SGK1 negatively regulates stress-activated signaling through inhibition of SEK1 function.

Notch interferes with the scaffold function of JNK-interacting protein 1 to inhibit the JNK signaling pathway.

The transmembrane protein Notch is cleaved by gamma-secretase to yield an active form, Notch intracellular domain (Notch-IC), in response to the binding of ligands, such as Jagged. Notch-IC contributes to the regulation of a variety of cellular events, including cell fate determination during embryonic development as well as cell growth, differentiation, and survival. We now show that Notch1-IC suppresses the scaffold activity of c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1) in the JNK signaling pathway. Notch1-IC physically associated with the JNK binding domain of JIP1 and thereby interfered with the interaction between JIP1 and JNK. JIP1 mediated the activation of JNK1 induced by glucose deprivation in mouse embryonic fibroblasts, and ectopic expression of Notch1-IC inhibited JNK activation and apoptosis triggered by glucose deprivation. Taken together, these findings suggest that Notch1-IC negatively regulates the JNK pathway by disrupting the scaffold function of JIP1.