Richa Tyagi

Rhes, a striatal-enriched small G protein, mediates mTOR signaling and L-DOPA–induced dyskinesia

L-DOPA–induced dyskinesia, the rate-limiting side effect in the therapy of Parkinsons disease, is mediated by activation of mammalian target of rapamycin (mTOR) signaling in the striatum. We found that Ras homolog enriched in striatum (Rhes), a striatal-specific protein, binds to and activates mTOR. Moreover, Rhes−/− mice showed reduced striatal mTOR signaling and diminished dyskinesia, but maintained motor improvement on L-DOPA treatment, suggesting a therapeutic benefit for Rhes-binding drugs.

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2.

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) cochaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks), of which IP6K2 has been implicated in p53-associated cell death. In the present study we report an apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex, thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine 15 to activate the cell death program in human cancer cells and in murine B cells.

Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

Antidepressant action of ketamine via mTOR is mediated by inhibition of nitrergic Rheb degradation

As traditional antidepressants act only after weeks/months, the discovery that ketamine, an antagonist of glutamate/N-methyl-d-aspartate (NMDA) receptors, elicits antidepressant actions in hours has been transformative. Its mechanism of action has been elusive, though enhanced mammalian target of rapamycin (mTOR) signaling is a major feature. We report a novel signaling pathway wherein NMDA receptor activation stimulates generation of nitric oxide (NO), which S-nitrosylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitrosylated GAPDH complexes with the ubiquitin-E3-ligase Siah1 and Rheb, a small G protein that activates mTOR. Siah1 degrades Rheb leading to reduced mTOR signaling, while ketamine, conversely, stabilizes Rheb that enhances mTOR signaling. Drugs selectively targeting components of this pathway may offer novel approaches to the treatment of depression.

Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction

Significance The induction of immediate early genes (IEGs) by neural stimuli underlies much of the plasticity of brain function, but regulatory mechanisms have been obscure. Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic enzyme that displays inositol phosphate kinase activity and phosphatidylinositol kinase activity and exhibits physiologically noncatalytic actions such as stabilizing the mammalian target of rapamycin complex 1 complex. We report that IPMK is required for IEG induction by neural activation and neurotrophic stimuli. We have elucidated the molecular mechanisms responsible for IPMK influences; namely, that it enhances the transcriptional coactivation ability of Creb-binding protein (CBP). This epigenetic regulation of IEGs may have both neural and nonneural implications, as IPMK and CBP are broadly expressed in a variety of tissues. Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

Significance Attachment of the small protein ubiquitin to other proteins, a process called “ubiquitylation,” triggers protein degradation. The Cullin and signalosome protein families cooperatively mediate this process, but how they are regulated has been obscure. We show that an inositol polyphosphate with seven phosphates, hence designated IP7, is critical. Under basal conditions, the enzyme that generates IP7 is bound to the Cullin/signalosome complex, which is thereby maintained in an inactive state. Stressful stimuli, such as UV radiation, stimulate the enzyme to form IP7, which dissociates the complex, leading to activation of the Cullins with attendant ubiquitylation and degradation of target proteins. This process may play a key role in how cells respond to environmental stressors. Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death.

Rheb Inhibits Protein Synthesis by Activating the PERK-eIF2α Signaling Cascade

Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK). Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

Huntington’s disease: Neural dysfunction linked to inositol polyphosphate multikinase

Significance Huntington’s disease (HD) is a progressive neurodegenerative disorder affecting the striatum. The striatal-enriched transcription factor COUP-TF-interacting protein 2 (Ctip2) is depleted in HD and has been identified as a putative transcription factor for the enzyme inositol polyphosphate multikinase (IPMK). IPMK displays soluble inositol phosphate kinase activity, lipid kinase activity, and several noncatalytic activities including its role as a transcriptional coactivator. We describe severe depletion in IPMK protein in HD patients and several animal and cell models of the disease. IPMK overexpression rescues the metabolic impairments in a cell model of HD. Furthermore, delivery of IPMK in a transgenic HD model improves pathological changes and motor performance. The Ctip2–IPMK–Akt signaling pathway provides a previously unidentified therapeutic target for HD. Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD.

Inositol polyphosphate multikinase is a coactivator for serum response factor-dependent induction of immediate early genes

Significance The inositol polyphosphate system is a major intracellular signaling modality. Inositol polyphosphate multikinase (IPMK) is essential for this system, converting inositol 1,4,5-trisphosphate (IP3) to IP4 and IP4 to IP5. IPMK also is a physiologic PI3-kinase. In a noncatalytic manner, IPMK stabilizes the mammalian target of rapamycin (mTOR) complex as well as tumor suppressor protein p53 and CREB-binding protein (CBP)/E1A binding protein p300. We now demonstrate that IPMK is a transcriptional coactivator for serum response factor (SRF) signaling, which is markedly reduced in IPMK-deleted mice and augmented by IPMK overexpression. IPMK binds directly to SRF, facilitating its interaction with serum response elements of multiple immediate early genes. Agents influencing IPMK interactions with SRF may be useful in regulating the SRF signaling system. Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic protein. It displays both inositol phosphate kinase and phosphatidylinositol kinase catalytic activities. Noncatalytically, IPMK stabilizes the mammalian target of rapamycin complex 1 and acts as a transcriptional coactivator for CREB-binding protein/E1A binding protein p300 and tumor suppressor protein p53. Serum response factor (SRF) is a major transcription factor for a wide range of immediate early genes. We report that IPMK, in a noncatalytic role, is a transcriptional coactivator for SRF mediating the transcription of immediate early genes. Stimulation by serum of many immediate early genes is greatly reduced by IPMK deletion. IPMK stimulates expression of these genes, an influence also displayed by catalytically inactive IPMK. IPMK acts by binding directly to SRF and thereby enhancing interactions of SRF with the serum response element of diverse genes.

Global IP6K1 deletion enhances temperature modulated energy expenditure which reduces carbohydrate and fat induced weight gain

Objective IP6 kinases (IP6Ks) regulate cell metabolism and survival. Mice with global (IP6K1-KO) or adipocyte-specific (AdKO) deletion of IP6K1 are protected from diet induced obesity (DIO) at ambient (23 °C) temperature. AdKO mice are lean primarily due to increased AMPK mediated thermogenic energy expenditure (EE). Thus, at thermoneutral (30 °C) temperature, high fat diet (HFD)-fed AdKO mice expend energy and gain body weight, similar to control mice. IP6K1 is ubiquitously expressed; thus, it is critical to determine to what extent the lean phenotype of global IP6K1-KO mice depends on environmental temperature. Furthermore, it is not known whether IP6K1 regulates AMPK mediated EE in cells, which do not express UCP1. Methods Q-NMR, GTT, food intake, EE, QRT-PCR, histology, mitochondrial oxygen consumption rate (OCR), fatty acid metabolism assays, and immunoblot studies were conducted in IP6K1-KO and WT mice or cells. Results Global IP6K1 deletion mediated enhancement in EE is impaired albeit not abolished at 30 °C. As a result, IP6K1-KO mice are protected from DIO, insulin resistance, and fatty liver even at 30 °C. Like AdKO, IP6K1-KO mice display enhanced adipose tissue browning. However, unlike AdKO mice, thermoneutrality only partly abolishes browning in IP6K1-KO mice. Cold (5 °C) exposure enhances carbohydrate expenditure, whereas 23 °C and 30 °C promote fat oxidation in HFD-KO mice. Furthermore, IP6K1 deletion diminishes cellular fat accumulation via activation of the AMPK signaling pathway. Conclusions Global deletion of IP6K1 ameliorates obesity and insulin resistance irrespective of the environmental temperature conditions, which strengthens its validity as an anti-obesity target.