Department of Cancer Biology, City of Hope, 1500 East Duarte Road, Duarte, CA 91107, USA; Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.
Department of Cancer Biology, City of Hope, 1500 East Duarte Road, Duarte, CA 91107, USA.
Cell Signal. 2017 Dec;40:53-61. doi: 10.1016/j.cellsig.2017.08.012. Epub 2017 Sep 1.
t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (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 and activation of protein kinase A (PKA). 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 overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDp), 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. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDp cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp's effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp's activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp-mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.
t-Darpp 是多巴胺和 cAMP 调节的 32kDa 磷蛋白（Darpp-32）的截断形式，已被证明通过持续的磷脂酰肌醇-4,5-二磷酸 3-激酶（PI3K）/Akt 途径信号传导和蛋白激酶 A（PKA）的激活来赋予曲妥珠单抗（一种针对 Her2 的抗癌药物）耐药性。t-Darpp 介导的 PKA 激活的机制尚不清楚。在 PKA 全酶中，当催化亚基与调节亚基 RI 或 RII 结合时，激酶活性受到抑制。我们研究了过表达 t-Darpp（SK.tDp）或 T39A 磷酸化突变体（SK.tDp）的细胞系以及空载体对照细胞系（SK.empty）中的 PKA 活性和全酶组成。我们还评估了这些细胞系中 t-Darpp 与 PKA 催化亚基（PKAc）或调节亚基 RI 和 RII 之间的蛋白-蛋白相互作用。SK.tDp 细胞具有升高的 PKA 活性，并且 RI 与 PKAc 的结合减少，而 SK.tDp 细胞没有这些特性。此外，野生型 t-Darpp 与 RI 结合。Darpp-32 的同时表达逆转了 t-Darpp 对 PKA 全酶状态的影响，这与早先的观察结果一致，即 Darpp-32 逆转了 t-Darpp 对 PKA 的激活。总之，t-Darpp 在 T39 处的磷酸化似乎对于 t-Darpp 介导的 PKA 激活至关重要，并且这种激活似乎是通过与 RI 结合和将 RI 从 PKAc 上隔离来发生的。t-Darpp-RI 相互作用可能是一种可药物靶向的靶点，可降低耐药性癌症中的 PKA 活性。
