MIT Libraries logoDSpace@MIT

MIT
View Item 
  • DSpace@MIT Home
  • MIT Open Access Articles
  • MIT Open Access Articles
  • View Item
  • DSpace@MIT Home
  • MIT Open Access Articles
  • MIT Open Access Articles
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

An epitope tag alters phosphoglycerate dehydrogenase structure and impairs ability to support cell proliferation

Author(s)
Kini, Mitali; Mattaini, Katherine Ruth; Davidson, Shawn Michael; Fiske, Brian Prescott; Vander Heiden, Matthew G.; Brignole, Edward J; Drennan, Catherine L; ... Show more Show less
Thumbnail
Download40170_2015_Article_131.pdf (3.137Mb)
OPEN_ACCESS_POLICY

Open Access Policy

Creative Commons Attribution-Noncommercial-Share Alike

Metadata
Show full item record
Abstract
Background The gene encoding the serine biosynthesis pathway enzyme PHGDH is located in a region of focal genomic copy number gain in human cancers. Cells with PHGDH amplification are dependent on enzyme expression for proliferation. However, dependence on increased PHGDH expression extends beyond production of serine alone, and further studies of PHGDH function are necessary to elucidate its role in cancer cells. These studies will require a physiologically relevant form of the enzyme for experiments using engineered cell lines and recombinant protein. Results The addition of an N-terminal epitope tag to PHGDH abolished the ability to support proliferation of PHGDH-amplified cells despite retention of some activity to convert 3-PG to PHP. Introducing an R236E mutation into PHGDH eliminates enzyme activity, and this catalytically inactive enzyme cannot support proliferation of PHGDH-dependent cells, arguing that canonical enzyme activity is required. Tagged and untagged PHGDH exhibit the same intracellular localization and ability to produce D-2-hydroxyglutarate (D-2HG), an error product of PHGDH, arguing that neither mislocalization nor loss of D-2HG production explains the inability of epitope-tagged PHGDH to support proliferation. To enable studies of PHGDH function, we report a method to purify recombinant PHGDH and found that untagged enzyme activity was greater than N-terminally tagged enzyme. Analysis of tagged and untagged PHGDH using size exclusion chromatography and electron microscopy found that an N-terminal epitope tag alters enzyme structure. Conclusions Purification of untagged recombinant PHGDH eliminates the need to use an epitope tag for enzyme studies. Furthermore, while tagged PHGDH retains some ability to convert 3PG to PHP, the structural alterations caused by including an epitope tag disrupts the ability of PHGDH to sustain cancer cell proliferation.
Date issued
2015-04
URI
http://hdl.handle.net/1721.1/97570
Department
Massachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Chemistry; Koch Institute for Integrative Cancer Research at MIT
Journal
Cancer & Metabolism
Publisher
BioMed Central
Citation
Mattaini, Katherine R, Edward J Brignole, Mitali Kini, Shawn M Davidson, Brian P Fiske, Catherine L Drennan, and Matthew G Vander Heiden. “An Epitope Tag Alters Phosphoglycerate Dehydrogenase Structure and Impairs Ability to Support Cell Proliferation.” Cancer Metab 3, no. 1 (April 29, 2015).
Version: Final published version
ISSN
2049-3002

Collections
  • MIT Open Access Articles

Browse

All of DSpaceCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

My Account

Login

Statistics

OA StatisticsStatistics by CountryStatistics by Department
MIT Libraries
PrivacyPermissionsAccessibilityContact us
MIT
Content created by the MIT Libraries, CC BY-NC unless otherwise noted. Notify us about copyright concerns.