t-Darpp is an elongated monomer that binds to calcium and is phosphorylated by cyclin-dependent kinases 1 and 5

ABSTRACT t-Darpp is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil structures. t-Darpp binds to calcium, but not to other metals commonly found in biological systems. The T39 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, blue native gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.