Abstract 3076: MEKK1 is a novel modulator of the Arf-p53 pathway via Dmp1 phosphorylation

Abstract

Abstract Dmp1 (cyclin D-interacting myb-like protein 1; Dmtf1) is a transcription factor isolated in yeast two-hybrid screen as a cyclin D2 binding partner. As a transcription factor, Dmp1 binds to nonameric DNA consensus sequences CCCG(G/T)ATG(T/C) to act as an activator or a repressor. Extensive molecular and genetic evidence links tumor suppressor function of Dmp1 to regulation of the ARF-p53 pathway. In fact, Dmp1 directly binds to p19Arf (p14ARF in humans) promoter and induces its expression. Increase in ARF level leads to protection of p53 from Mdm2-mediated degradation and subsequent increase in p53 target genes involved in cell cycle arrest and apoptosis. Oncogenic Ras and Her2/neu induce Dmp1 expression leading to Arf-, p53-dependent cell cycle arrest. Human DMP1 locus is hemizygously deleted in ∼50% of breast carcinomas, which is mutually exclusive of ARF and p53 loss. Based on the amino acid sequence, predicted molecular weight of Dmp1 is 85kDa. However, Dmp1 migrates to ∼120-130kDa on a SDS-PAGE gel, suggesting post-translational modifications. Although signal transduction pathways regulating Dmp1 transcripton have been studied, the role of Dmp1 protein modification is unknown. Treatment of Dmp1 protein with calf intestine phosphatase and running a 2D SDS-PAGE gel, we have shown that one mode of post-translational modification is via phosphorylation. The presence of phosphates on several Serines and/or Threonines was confirmed with mass spectrometry. Using an Arf luciferase reporter assay in 3T3 cells, we found that MEKK1 (MEK kinase 1) synergizes with Dmp1 on the Arf promoter activity, with the synergy attenuated when several of the putative phosphorylation sites on Dmp1 were mutated. The effect observed was Dmp1-dependent since MEKK1 was unable to activate the Arf promoter in Dmp1-null cells. Full length (inactive) MEKK1 was unable to synergize with Dmp1 on the Arf promoter activity suggesting necessity of the kinase activity. The ability of MEKK1 to directly phosphorylate Dmp1 was confirmed in in vitro kinase assay using purified MEKK1 and Flag-Dmp1 proteins and Arf reporter assays where known kinases downstream of MEKK1(MEK1, MEK4/7, JNK1/2) were inhibited or knocked down. MEKK1 and Dmp1 co-immunoprecipitated when expressed in 3T3 cells. To study the effect of MEKK1 on endogenous Dmp1 and Arf, we either expressed MEKK1 or treated cells with MEKK1-activating drugs, cisplatin and etoposide. In this setting, endogenous p14ARF was induced and the banding pattern of Dmp1 protein was shifted in a SDS-PAGE gel indicating protein modification. Here we show that Dmp1 can be directly phosphorylated by MEKK1 to modulate Dmp1 transcriptional activity on the Arf promoter. Therefore, MEKK1 may activate the Arf-p53 pathway via direct Dmp1 protein phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3076. doi:10.1158/1538-7445.AM2011-3076