Phosphorylation of CEACAM1 by Calmodulin Kinase IID in a 3D Model of Mammary Gland Lumen Formation [Signal Transduction]

December 3rd, 2013 by Nguyen, T., Chen, C.-J., Shively, J. E.

Carcinoembryonic antigen related cell adhesion molecule-1 (CEACAM1), a transmembrane protein, expressed on normal breast epithelial cells is down-regulated in breast cancer. Phosphorylation of Thr-457 on the short cytoplasmic domain isoform (CEACAM1-SF) that is predominant in normal epithelial cells is required for lumen formation in a 3D model that involves apoptosis of the central acinar cells (Kirshner et al. 100:521-6, 2003). Calmodulin kinase IID (CaMKIID) was selected as a candidate for the kinase required for Thr-457 phosphorylation from a gene chip analysis comparing genes upregulated in MCF7 cells expressing wild type CEACAM1-SF compared to the T457A mutated gene (Chen et al., J. Biol. Chem. 282:5749-60, 2007). Upregulation of CaMKIID during lumen formation was confirmed by analysis of mRNA and protein levels. CaMKIID was able to phosphorylate a synthetic peptide corresponding to the cytoplasmic domain of CEACAM1-SF and was covalently bound to biotinylated and T457C modified peptide in the presence of a kinase trap previously described by Shokat and coworkers (J. Am. Chem. Soc. 126: 9160-1, 2004). When cell lysates from wild type transfected MCF7 cells undergoing lumen formation were incubated with the peptide and kinase trap, a cross-linked band corresponding to CaMKIID was observed. When these cells were treated with an RNAi that inhibits CaMKIID expression, lumen formation was blocked by over 90%. We conclude that CaMKIID specifically phosphorylates Thr-457 on CEACAM1-SF, which in turn, regulates the process of lumen formation via apoptosis of the central acinar cells.