Abstract 2128: Phosphorylation of Id2 at the N-terminus modulates Id2 degradation and mediates cell cycle regulation in neural progenitor cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Glioblastoma is the most common and aggressive type of primary brain tumor in adults with more than 14,000 new cases diagnosed each year in the US. Although surgical resection, radiation, and cytotoxic therapies are available these current treatment methods are not curative and the median survival of patients remains at 12-15 months. Inhibitor of DNA binding proteins (Id1-Id4) are a family of genetically encoded dominant negative regulators of basic helix-loop-helix (bHLH) transcription factors. Id proteins are widely reported to inhibit differentiation and promote cell cycle transit in neural progenitor cells (NPCs) and have been implicated in the development of glioma. Our laboratory has observed a poor correlation between Id2 mRNA levels and Id2 protein levels in multiple cell types which led us to seek post-translational modifications that effected steady state Id2 protein levels and key Id2 mediated cellular functions. Using mass spectrometry we have identified three phosphorylation sites within the N-terminus of Id2 in proliferating NPCs. To interrogate the importance of Id2 N-terminal phosphorylation, Id2-/- NPCs were modified to express WT Id2 or various Id2 mutants expressing proteins that could not be phosphorylated at the N-terminus. We observed that NPCs expressing these mutants had higher steady state levels of Id2 than NPCs expressing WT Id2. It is known that WT Id2 is rapidly degraded by the proteasome. However, when proliferating NPCs were treated with cyclohexamide, phospho-ablated Id2 molecules exhibited a longer half-life than WT Id2 molecules indicating that loss of N-terminal phosphorylation results in resistance to proteasome-mediated degradation. Moreover, NPCs expressing this degradation-resistant, phospho-ablated Id2 protein proliferate more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Seeking to identify molecules whose inhibition might enhance Id2 phosphorylation, we evaluated the activity of multiple phosphatase inhibitors and identified phosphatases that could potentially function to stabilize pro-proliferative Id2 in NPCs. Calyculin A treatment caused a significant loss of Id2 protein suggesting that the PP1, PP2A, PP4 and/or PP6 phosphatases were likely regulators of Id2. To complement these pharmacologic studies we used a genetic approach to decrease PP2A expression and found that Id2 protein levels were decreased. Our findings indicate that inhibition of this phosphatase may provide a novel mechanism to decrease Id2 protein levels by causing rapid degradation of Id2. Citation Format: Jaclyn Sullivan, Matthew Havrda, Brenton Paolella, Arminja Kettenbach, Scott Gerber, Mark A. Israel. Phosphorylation of Id2 at the N-terminus modulates Id2 degradation and mediates cell cycle regulation in neural progenitor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2128. doi:10.1158/1538-7445.AM2015-2128