A single nucleotide polymorphism in steroid receptor coactivator 1 diminishes ERa coactivation.
Authors: AS Richter, S Oesterreich
Journal: Cancer Research (2009)

Abstract Abstract #605 Background: Single nucleotide polymorphisms (SNPs) can contribute to alterations in treatment efficiency. The 3814C&amp;gt;T SNP that causes a P1272S amino acid change located in the activation domain-2 (AD2) of the steroid receptor coactivator SRC-1, has been shown to be associated with decreased bone mineral density in women undergoing tamoxifen treatment for breast cancer. It is well described that members of the SRC family are regulated by posttranslational modifications. Phosphorylation has been shown to be critical for their activity. It is known that histone-acetyl-transferase (HAT) activity of SRC proteins can be controlled through phosphorylation. The C-terminus of SRC-1 is one of the major region to be phosphorylated, possibly to perform proper HAT activity. Therefore, a change from proline to serine at position 1272 is likely to have impact on phosphorylation and subsequent HAT activity. Additionally, in silico analysis of the sequence suggests that the variant removes a potential Glycogen synthase kinase 3 (GSK3) target motif at amino acid 1275. Intriguingly, phosphorylation by GSK3 has been shown to be involved in modulating the activity and functional lifetime of another member of the SRC family, SRC-3.&amp;#x2028; Methods: In silico analysis to identify changes in phosphorylation sites after introducing the SNP was performed using http://www.cbs.dtu.dk/services/NetPhos. Mutants P1272S, P1272A, P1272E were generated by mutagenesis using QuickChange® Site-directed Mutagenesis Kit following the manufacturer's protocol. ERE-Luciferase reporter assays were conducted in HEK293 cells to compare the ability of SRC-1 wildtype (WT), variant P1272S, and mutants P1272A or P1272E to activate ERα-mediated transcription. Protein levels were determined after immunoblotting.&amp;#x2028; Results: Post-translational modifications, particularly in the form of phosphorylation, are critical for the activity of members from the p160 family of coactivators as described above. Thus, we examined if the introduction of P1272S induces or destroys any phosphorylation sites within SRC-1. We identified phosphorylation sites by scoring the SRC-1 protein sequence including proline (WT) versus serine (SNP). Indeed we found that a cdc2 phosphorylation site is created and a cdk5 phosphorylation site is partially destroyed when serine is present instead of proline. Using an ERE-Luciferase reporter assay in HEK293 cells, we observed a significant loss of co-activation by the variant compared to SRC-1 WT. Transfecting P1272E showed a similar decrease to P1272S in these assays, suggesting the effect of decreased coactivation might be due to the introduction of a new phosphorylation site. Interestingly, transfecting P1272A was not able to rescue the phenotype of P1272S. This finding suggests an additional role for changes in protein folding with subsequent changes in protein activity. Furthermore, immunoblotting of SRC-1 WT or P1272S after estrogen treatment revealed a stabilizing effect of SRC-1 WT but not for the SNP. This further implies that introducing this SNP results in a decreased protein level and subsequently in decreased activity after hormone treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 605.