Rahman, Hazir

The mammalian target of rapamycin (mTOR) is an            intracellular protein kinase that plays a key role in            the control of cell growth, differentiation, cell            survival, and cell proliferation. In humans, mTOR            deregulation is implicated in parasitic, fungal,            bacterial and viral infections, immune disorders,            diabetes, obesity, cardiac diseases, renal            abnormalities, and various cancers. mTOR was first            identified as TOR in Saccharomyces cerevisiae. It            exists in two structurally and functionally distinct            complexes, mTOR complex 1 (mTORC1), and mTOR complex 2            (mTORC2). mTORC1, a rapamycin sensitive protein            complex, senses the availability of growth factors,            nutrients, cellular energy levels, and is actively            involved in cellular transcription and translation            processes. mTORC1 performs a range of biological            functions with the help of its interacting proteins.            These interacting proteins act as scaffolds and recruit            substrates and regulatory proteins required for mTOR            kinase function. The present study was undertaken to            identify novel interacting partners of mTORC1 that            specifically interact with mTORC1 to enable this            crucial cell signaling hub to carry out its biological            functions.Human T cells (CCRF-CEM) and human embryonic kidney            (HEK293) cell lines were used to identify novel            interacting partners of mTORC1. Endogenous mTORC1 along            with its interacting proteins were purified using            raptor monoclonal antibodies and immunoblotted to            confirm the mTORC1 specific purification. Following            confirmation of mTORC1 specific purification by            immunoblotting, the remaining IP elutes were resolved            on SDS-PAGE and stained with silver nitrate. Protein            bands from the gel were excised, processed by in-gel            digestion and identified by nano-LC ESI Q-TOF MS/MS            analysis. The mass spectrometric identification of            endogenous mTORC1 interacting proteins was further            validated by expressing the myc-tag raptor pRK5 vector            in CCRF-CEM and HEK293 cells. Myc-tag raptor component            of mTORC1 was isolated by pulled-down from the cell            lysate using an affinity column and conjugated            monoclonal myc-tag antibodies using agarose beads. The            co-purified elutes were resolved on SDS-PAGE and mTORC1            specific purification was first confirmed by            immunoblotting, and later identified by nano-LC ESI            Q-TOF MS/MS analysis. A total of 10 novel interacting            proteins (hnRNP A2/B1, SRSF7, RP-P0, NCL, DNM2, GAPDH,            2-OADH, GLT25D1, PHB2, Edc4) were identified in both            endogenous and myc-tag mTORC1 purifications. Functional            annotation analysis demonstrated that these ten            proteins are involved in important biological            functions. Three proteins, hnRNP A2/B1, SRSF7, and            Edc4, are important for mRNA processing while two            proteins, RP-P0 and NCL, were involved in transcription            and translation. One protein, DNM2, identified in            mTORC1 specific purifications, is associated with            intracellular trafficking, while two proteins, GAPDH            and 2-OADH, are involved in carbohydrate metabolism.            Moreover, two proteins, GLT25D1 and PHB2, are involved            in post-translation modification, protein turnover and            chaperone functions. The mass spectrometric            identification of Edc4, DNM2, and hnRNP A2/B1 proteins            were further confirmed by immunoblotting using protein            specific antibodies.Enhancer of mRNA decapping protein 4 (Edc4) was            consistently identified as a new interacting protein            with mTORC1 in both the endogenous and myc-tag raptor            component of mTORC1. Edc4 has a suggested role in mRNA            decapping and repression of miRNA mediated translation.            The potential interaction of Edc4 with mTORC1 was            further confirmed using reverse co-immunoprecipitation.            Quantitative co-localization using confocal microscopy            demonstrated a high degree of pixel overlapping between            Edc4 protein with raptor component of mTORC1 both            inside and outside of P bodies. Incubation of cells            under leucine starved conditions increased the total            expression of Edc4. Leucine is an essential amino acid            which is a positive regulator of mTORC1 kinase            activity, providing evidence that mTORC1 may be            involved in the regulation of Edc4. Furthermore,            rapamycin increased total Edc4 protein expression but            at the same time decreased the Edc4 interaction with            mTORC1, further evidence of mTORC1 involvement in Edc4            regulation. We further examined the effects of            rapamycin on Edc4 phosphorylation status. Rapamycin            treatment resulted in a significant decrease in total            serine phosphorylated Edc4 protein signal, suggesting            the involvemnt of mTORC1 kinase activity in the            regulation of Edc4. In addition, we observed that            rapamycin significantly decreased the total 5´-capped            mRNA. These findings suggest that mTORC1, by            interacting with Edc4, inactivates the Edc4 through            serine phosphorylation, and regulates its expression.            This results in hyper-phosphorylated Edc4 which is then            no longer available for mRNA decapping activity. The            inhibition of mTORC1 by rapamycin results in an            increased amount of dephosphorylated Edc4, and            consequently higher cellular decapping activity, and            less total 5´-capped mRNA in the cell. These findings            provide the first evidence for the pivotal role of            mTORC1 in Edc4 regulation. Further in-depth studies are            required to get a complete understanding of the            biological interplay of mTORC1 signaling in the mRNA            decapping process. Functional characterization of these            novel interacting proteins may be helpful in            understanding the complexity of the mTORC1 network and            may offer new targets for therapeutic interventions in            human diseases associated with deregulated mTORC1            signaling

Georg-August-University Göttingen

Identification of interacting partners of mammalian target of rapamycin complex 1 (mTORC1) assembly in human lymphocytes

https://ediss.uni-goettingen.de/bitstream/11858/00-1735-0000-0006-AE46-6/1/rahman.pdf

Identification of interacting partners of mammalian target of rapamycin complex 1 (mTORC1) assembly in human lymphocytes

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Georg-August-University Göttingen