Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway. t-Darpp over-expression results in enhanced activity of protein kinase A (PKA), but the mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines that over-express t-Darpp (SK.tDrp) or a T75A phosphorylation mutant (SK.tDrp.T75A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. Whereas SK.tDrp cells had elevated PKA activity, SK.tDrp.T75A cells did not. In agreement with the effects on activity, a proximity ligation assay showed that SK.tDrp cells, but not SK.tDrp.T75A cells, had diminished association of RI with PKAc. Moreover, t-Darpp was seen to bind directly to RI in co-immunoprecipitation studies. Cells that express both Darpp-32 and t-Darpp had PKA activity levels and RI-PKAc association that were similar to control SK.empty cells. This is consistent with our published findings that Darpp-32 reverses t-Darpp’s effects on trastuzumab resistance and CREB DNA binding activity (downstream of PKA). Taken together, our data suggest that T75 phosphorylation is crucial for t-Darpp-mediated PKA activation and this activation appears to occur through direct binding to RI and release of RI from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.</jats:p>
<jats:p>Citation Format: Dirk Theile, Shuhui Geng, Erin Denny, Jamil Momand, Susan E. Kane. t-Darpp stimulates protein kinase A activity by interacting with its RI regulatory subunit [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 4093. doi:10.1158/1538-7445.AM2017-4093</jats:p>