Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Background: We have previously shown that levels of the 32kDa dopamine and cAMP-regulated phosphoprotein Darpp-32 (Dp32) and its amino-truncated isoform t-Darpp (tDp) are altered in trastuzumab-resistant HER2+ breast cancer cells. Specifically, tDp is upregulated in models of trastuzumab resistance and over-expression of the protein is sufficient to confer trastuzumab resistance. Dp32 antagonizes the effect of tDp, exhibiting overall growth inhibitory properties in breast cancer cell lines. In neuronal cells, Dp32 is regulated by phosphorylation at two key threonine residues, T34 and T75. The T34 site is present only in Dp23 as tDp is truncated by the first 39 amino acids. The latter site is phosphorylated predominantly by cdk5 in vivo and by either cdk5 or cdk1 in vitro. It is not known whether tDp is similarly phosphorylated or if the same kinases act in breast cancer cells as in neuronal cells. Because we have preliminary data suggesting an interaction between tDp and cdk1 (but not cdk5) in HER2+ breast cancer cells, we investigated whether cdk1 or cdk5 could phosphorylate tDp at the T75 site.</jats:p>
<jats:p>Methods: We performed in vitro kinase assays using recombinant tDp and commercial cdk1 and cdk5 enzymes. Phosphorylation was also examined in cells that overexpress endogenous tDp (SK/HerR and BT/HerR cells), that overexpress exogenous tDp (SK.tDp cells) or that express exogenous tDp and Dp32 (SK.dDp). Cells were treated with either RO-3306, a cdk1 inhibitor, or Roscovitine, a pan-cdk inhibitor with preference for cdk5, and assayed by Western blotting for T75 phosphorylation. All experiments were repeated three times and calculated for statistical significance.</jats:p>
<jats:p>Results: In vitro kinase assays demonstrated that both cdk1 and cdk5 were capable of phosphorylating tDp at the T75 residue, with an apparent preferential activity by cdk1. In cell lines, RO-3306 led to decreased T75 phosphorylation within one hour. The effect of RO-3306 was also confirmed in a dose-dependent manner with optimal inhibition at a concentration of 10 μM. Roscovitine also led to decreased T75 phosphorylation at a concentration of 20 μM. In cells that express both Dp32 and tDp, the cdk inhibitors led to decreased phosphorylation of both proteins to similar extents.</jats:p>
<jats:p>Conclusion: Our study provides new insight into cdk1-mediated regulation of tDp and Dp32. We demonstrate that both cdk1 and cdk5 are able to phosphorylate these proteins but that cdk1 appears to be the predominant mediator of phosphorylation in breast cancer cell lines. A possible competition might occur between Dp32 and tDp, given their similar propensity for phosphorylation by cdk1. Further examination of the relationship between the two kinases and their activities toward tDp and Dp32 will help us delineate the mechanism by which tDp mediates trastuzumab resistance and Dp32 antagonizes this phenotype.</jats:p>
<jats:p>Citation Format: Erin Denny, Arianna Celis, Jamil Momand, Susan E. Kane. t-Darpp is phosphorylated by cdk1 and cdk5 in HER2+ breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A058.</jats:p>