Abstract 5866: t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites

Abstract

Abstract t-Darpp, one of two protein isoforms encoded by the PPP1R1B gene, is overexpressed in gastric, esophageal, colon, prostate, and breast cancers; and confers resistance to trastuzumab in Her2+ breast cancer cells. To gain insight into the trastuzumab-resistance mechanism of t-Darpp, we studied its structure, oligomerization status, metal-binding properties, and sites of phosphorylation. Circular dichroism spectroscopy analysis showed that recombinant t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil character at 25°C. Upon mild heat treatment (50°C for five min) the secondary structure does not appreciably change. Metal ion-binding analyses through inductively coupled plasma-mass spectrometry and graphite furnace-atomic absorption indicated that t-Darpp co-purifies with calcium, but not other metals commonly found in biological systems. The T75 site, critical for t-Darpp activation of the Akt signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, native blue gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments showed that the majority of t-Darpp (calculated mass = 19 kDa) exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a relatively large Stokes radius of 4.4 nm, suggesting an elongated structure. In summary, this study indicates that t-Darpp is an elongated, mild-heat-stable, and monomeric calcium-binding protein that is capable of being phosphorylated at T75 by CDK1 and CDK5. Blockage of t-Darpp calcium binding capacity or T75 phosphorylation may therefore help sustain Her2+ breast cancer sensitivity to trastuzumab therapy. Citation Format: Patrycja Magdziarz, You Feng, Dianlu Jiang, Elizabeth Parga, Arianna Celis, Erin Denny, Xiaoying Wang, Martin Phillips, Feimeng Zhou, Susan E. Kane, Jamil Momand. t-Darpp chemoresistence protein: insights into its structure, oligomerization status, calcium-binding properties and phosphorylation sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5866. doi:10.1158/1538-7445.AM2017-5866