Defining the Post-Translational Ubiquitination Landscape of PID1
Poster #: 148
Session/Time: A
Author:
Neel C. Herfarth, BS, MS
Mentor:
Anat Epstein, MD, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
The Epstein lab has shown that Phosphotyrosine Interaction Domain-containing protein 1 (PID1/NYGGF4) has inhibitory effects in malignant brain tumors. Consistent with this, they also showed that higher PID1 mRNA levels were correlated with better outcomes in patients with medulloblastoma (MB) and glioblastoma (GBM). Cisplatin, a chemotherapy used in therapy of some brain tumors, diminished PID1 protein in a manner that was inhibitable by bortezomib, a proteasome inhibitor. Consistent with this, they found that PID1 has a short half-life of 1-2 hours. In my summer project, I hypothesized that PID1 undergoes ubiquitination and is subject to proteasomal degradation.
METHODS:
HEK293T cells were transiently transfected with PID1 fusion proteins V5.PID1 or mEGFP.PID1 or their controls in RDAV vector, together with HA-tagged ubiquitin (HA-Ub) or HA-tagged small ubiquitin-like modifier (HA-SUMO). In some conditions, cells were treated with 100 nM bortezomib or vehicle for four hours. Cytoplasmic lysates were collected and subjected to anti-HA co-immunoprecipitation (co-IP) using anti-HA-affinity beads or IgG controls. Eluted proteins and the originating lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed by western blotting using anti-PID1 and anti-HA antibodies.
RESULTS:
Lanes containing eluates of anti-HA IP of cells expressing one of the PID1 fusion proteins, V5.PID1, or mEGFP.PID1 showed a ladder of anti-PID1-reactive bands when co-expressing HA-Ub, but not those co-expressing control non-PID1 plasmids and/or non-Ub HA. This indicates that PID1 is poly-ubiquitinated. This laddering was not seen in cells co-expressing PID1 and HA-SUMO, strengthening the conclusion that PID1 undergoes ubiquitination rather than SUMOylation. Finally, treatment with the proteasome inhibitor bortezomib increased the signal intensity of the PID1 laddering in HA-IPs, supporting that ubiquitinated PID1 undergoes degradation by the proteasome.
CONCLUSION:
These experiments demonstrate that PID1 is ubiquitinated, and the amount of laddered PID1 increases in the presence of proteasomal inhibition. This supports the idea that the amount of PID1 protein is regulated, at least in part, through the ubiquitin-proteasome system. Next experiments will examine if this also occurs in brain tumor-derived cell lines and whether endogenous PID1 is also subject to the ubiquitin-proteasome degradation system.
The Epstein lab has shown that Phosphotyrosine Interaction Domain-containing protein 1 (PID1/NYGGF4) has inhibitory effects in malignant brain tumors. Consistent with this, they also showed that higher PID1 mRNA levels were correlated with better outcomes in patients with medulloblastoma (MB) and glioblastoma (GBM). Cisplatin, a chemotherapy used in therapy of some brain tumors, diminished PID1 protein in a manner that was inhibitable by bortezomib, a proteasome inhibitor. Consistent with this, they found that PID1 has a short half-life of 1-2 hours. In my summer project, I hypothesized that PID1 undergoes ubiquitination and is subject to proteasomal degradation.
METHODS:
HEK293T cells were transiently transfected with PID1 fusion proteins V5.PID1 or mEGFP.PID1 or their controls in RDAV vector, together with HA-tagged ubiquitin (HA-Ub) or HA-tagged small ubiquitin-like modifier (HA-SUMO). In some conditions, cells were treated with 100 nM bortezomib or vehicle for four hours. Cytoplasmic lysates were collected and subjected to anti-HA co-immunoprecipitation (co-IP) using anti-HA-affinity beads or IgG controls. Eluted proteins and the originating lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed by western blotting using anti-PID1 and anti-HA antibodies.
RESULTS:
Lanes containing eluates of anti-HA IP of cells expressing one of the PID1 fusion proteins, V5.PID1, or mEGFP.PID1 showed a ladder of anti-PID1-reactive bands when co-expressing HA-Ub, but not those co-expressing control non-PID1 plasmids and/or non-Ub HA. This indicates that PID1 is poly-ubiquitinated. This laddering was not seen in cells co-expressing PID1 and HA-SUMO, strengthening the conclusion that PID1 undergoes ubiquitination rather than SUMOylation. Finally, treatment with the proteasome inhibitor bortezomib increased the signal intensity of the PID1 laddering in HA-IPs, supporting that ubiquitinated PID1 undergoes degradation by the proteasome.
CONCLUSION:
These experiments demonstrate that PID1 is ubiquitinated, and the amount of laddered PID1 increases in the presence of proteasomal inhibition. This supports the idea that the amount of PID1 protein is regulated, at least in part, through the ubiquitin-proteasome system. Next experiments will examine if this also occurs in brain tumor-derived cell lines and whether endogenous PID1 is also subject to the ubiquitin-proteasome degradation system.