PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling.

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.

MIP-T3 is a negative regulator of innate type I IFN response.

TNFR-associated factor (TRAF) 3 is an important adaptor that transmits upstream activation signals to protein kinases that phosphorylate transcription factors to induce the production of type I IFNs, the important effectors in innate antiviral immune response. MIP-T3 interacts specifically with TRAF3, but its function in innate IFN response remains unclear. In this study, we demonstrated a negative regulatory role of MIP-T3 in type I IFN production. Overexpression of MIP-T3 inhibited RIG-I-, MDA5-, VISA-, TBK1-, and IKKε-induced transcriptional activity mediated by IFN-stimulated response elements and IFN-ß promoter. MIP-T3 interacted with TRAF3 and perturbed in a dose-dependent manner the formation of functional complexes of TRAF3 with VISA, TBK1, IKKε, and IFN regulatory factor 3. Consistent with this finding, retinoic acid-inducible gene I- and TBK1-induced phosphorylation of IFN regulatory factor 3 was significantly diminished when MIP-T3 was overexpressed. Depletion of MIP-T3 facilitated Sendai virus-induced activation of IFN production and attenuated the replication of vesicular stomatitis virus. In addition, MIP-T3 was found to be dissociated from TRAF3 during the course of Sendai virus infection. Our findings suggest that MIP-T3 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors.

The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response.

RIG-I, a virus sensor that triggers innate antiviral response, is a DExD/H box RNA helicase bearing structural similarity with Dicer, an RNase III-type nuclease that mediates RNA interference. Dicer requires double-stranded RNA-binding protein partners, such as PACT, for optimal activity. Here we show that PACT physically binds to the C-terminal repression domain of RIG-I and potently stimulates RIG-I-induced type I interferon production. PACT potentiates the activation of RIG-I by poly(I:C) of intermediate length. PACT also cooperates with RIG-I to sustain the activation of antiviral defense. Depletion of PACT substantially attenuates viral induction of interferons. The activation of RIG-I by PACT does not require double-stranded RNA-dependent protein kinase or Dicer, but is mediated by a direct interaction that leads to stimulation of its ATPase activity. Our findings reveal PACT as an important component in initiating and sustaining the RIG-I-dependent antiviral response.

Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins.

OBJECTIVE: Mixed lineage leukemia (MLL) gene rearrangement is commonly observed in human leukemias. Many of the resultant MLL fusion proteins are found correlated with Ras signaling. Nevertheless, Ras mutations have only been reported in a small subset of MLL-rearranged leukemia. With the potential of developing new therapeutic regimens targeting Ras signaling pathway, we studied the role of MLL-AF4 family fusions and MLL-septin family fusions in the activation of Ras signaling in leukemogenesis. MATERIALS AND METHODS: Elk-1-driven luciferase reporter system was used to study the role of MLL-AF4, MLL-AF5q31, MLL-LAF4, MLL-CDCrel, MLL-MSF, and MLL-Septin 6 in the activation of Ras signaling. Dominant negative Ras S17N mutant and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 were employed to demonstrate the involvement of Ras and MEK in this transactivation event. The activation of endogenous Ras/MEK signaling pathway by MLL fusion proteins in leukemia cell lines was also addressed by immunoblot analysis and small interfering RNA knockdown approach. RESULTS: We demonstrated that MLL-AF4, MLL-AF5q31, and MLL-LAF4 activated Elk-1 transcription factor, one of the major downstream effectors of Ras. This activation was abolished in the presence of dominant negative Ras or MEK inhibitor U0126, indicating the requirements of Ras and MEK. We further showed that endogenous MEK is phosphorylated in a MLL-AF4-expressing leukemia cell line, whereas depletion of MLL-AF4 by small interfering RNA reduced the phospho-MEK level. CONCLUSION: Our findings suggest that MLL-AF4 family fusion oncoproteins can activate Elk-1 through Ras/MEK/extracellular signal-regulated kinase (ERK) pathway and strongly support the role of Ras signaling in the pathogenesis of MLL-rearranged leukemia.

Severe acute respiratory syndrome coronavirus M protein inhibits type I interferon production by impeding the formation of TRAF3.TANK.TBK1/IKKepsilon complex.

Severe acute respiratory syndrome (SARS) coronavirus is highly pathogenic in humans and evades innate immunity at multiple levels. It has evolved various strategies to counteract the production and action of type I interferons, which mobilize the front-line defense against viral infection. In this study we demonstrate that SARS coronavirus M protein inhibits gene transcription of type I interferons. M protein potently antagonizes the activation of interferon-stimulated response element-dependent transcription by double-stranded RNA, RIG-I, MDA5, TBK1, IKKepsilon, and virus-induced signaling adaptor (VISA) but has no influence on the transcriptional activity of this element when IRF3 or IRF7 is overexpressed. M protein physically associates with RIG-I, TBK1, IKKepsilon, and TRAF3 and likely sequesters some of them in membrane-associated cytoplasmic compartments. Consequently, the expression of M protein prevents the formation of TRAF3.TANK.TBK1/IKKepsilon complex and thereby inhibits TBK1/IKKepsilon-dependent activation of IRF3/IRF7 transcription factors. Taken together, our findings reveal a new mechanism by which SARS coronavirus circumvents the production of type I interferons.

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA.

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.