Regulation of mTORC1 by phosphatidic acid: mechanism and structural insight

Rosenberger, Christina Laura

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https://hdl.handle.net/2142/92925 Copy

Description

Title

Regulation of mTORC1 by phosphatidic acid: mechanism and structural insight

Author(s)

Rosenberger, Christina Laura

Issue Date

2016-07-07

Director of Research (if dissertation) or Advisor (if thesis)

Chen, Jie

Doctoral Committee Chair(s)

Chen, Jie

Committee Member(s)

• Belmont, Andrew
• Chen, Lin-Feng
• Rivier, David

Department of Study

Cell & Developmental Biology

Discipline

Cell and Developmental Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Mammalian target of rapamycin (mTOR)
• phosphatidic acid
• phospholipase D (PLD)
• DEP Domain Containing MTOR-Interacting Protein (DEPTOR)
• hyperactive
• cancer

Abstract

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase with remarkable control over cellular status. As a master regulator, mTOR integrates a variety of intra- and extra-cellular signals in order to coordinate them with appropriate gene expression, protein synthesis, metabolism, cell migration, autophagy – the list goes on! mTOR signaling, with involvement in so many important cellular processes, can have detrimental physiological effects when dysregulated. Aberrant mTOR signaling is now known to contribute to a great number of the major pathologies we face today. Understanding how mTOR is normally regulated is, therefore, important for informing the development of effective and specific therapeutics for diseases like cancer and diabetes. Significant research efforts over the last two decades have informed our current understanding of the extensive mTOR signaling pathways, but many questions remain. In my dissertation work I have investigated longstanding mysteries of mTOR activation by mitogens and nutrients, with specific focus on the mechanism by which the lipid second messenger, phosphatidic acid, activates mTOR complex 1. The mammalian target of rapamycin complex 1 (mTORC1) is regulated, in part, by the endogenous inhibitor DEPTOR. However, the mechanism of DEPTOR regulation with regard to rapid mTORC1 activation remains unknown. In collaboration with Dr. Mee Sup Yoon, I discovered that DEPTOR is rapidly and temporarily dissociated from mTORC1 upon mitogenic stimulation. We demonstrated that this mitogen stimulated DEPTOR dissociation is blocked by inhibition or depletion of the mTORC1 regulator, phospholipase D (PLD), and is recapitulated with the addition of the PLD product phosphatidic acid (PA). Parallel mass spectrometry analysis independently identified DEPTOR as an mTOR binding partner dissociated by PA. Interestingly, I found that only PA species with unsaturated fatty acid chains, such as those produced by PLD, are capable of displacing DEPTOR and activating mTORC1, with high affinity for the FRB domain of mTOR. Our findings, detailed in Chapter II reveal a mechanism of acute mTORC1 regulation that was previously unidentified and provide a molecular explanation for the exquisite specificity of PA function. In light of PA’s essential role in mTORC1 activation, I found it striking that mTOR proteins containing one of several point mutations have been reported to remain catalytically active in conditions when amino acids, and therefore PA, are absent. The existence of such hyperactive mTOR prompted me to ask whether a point mutation can render mTOR independent of regulation by PA and, if so, by what mechanism? In Chapter III, I describe how by examining the activity of several mTOR proteins each carrying a unique point mutation known to be associated with human cancer, I discovered that individual point mutations can confer varying degrees of PA-independent mTORC1 activity. My finding that an L1460P mutation in mTOR's FAT domain, S2215Y mutation in the kinase N-lobe, and E2419K in the kinase C-lobe all confer some mTORC1 activity in the absence of PA suggests that it is possible for PA-independent mTORC1 activity to result by more than one mechanism. Having identified that the activity of S2215Y mTOR is especially independent of PA and noting the implications of S2215Y for mTORC1’s structure, I propose that control of catalytic cleft access is a major aspect of mTORC1 activation by PA. My investigation also produced striking evidence that mTOR autophosphorylation, which is significantly hyperactivated by the R2505P mutation, does not necessarily correlate with PA-mediated mTORC1 phosphorylation of canonical substrates. Taken together, the experiments detailed in this chapter provide insight into the mechanism of mTORC1 activation by PA and carry significant therapeutic implications. Lastly, in Appendix A, I document several of my preliminary investigations of localization-dependent regulation of mTOR signaling. I have observed that both mTOR and raptor have a nuclear presence and that the proteins can localize there independently of each other. Additionally, I present evidence that nuclear mTORC1 phosphorylates the transcriptional repressor, Maf1, in a manner independent of the canonical mTORC1 pathway. Finally, I report for the first time that unsaturated PA species appear to recruit mTORC2 to detergent-sensitive cellular regions that I believe may be mitochondria-associated membranes.

Graduation Semester

2016-08

Type of Resource

text

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Copyright and License Information

Copyright 2016 Christina Laura Rosenberger

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