Abstract
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 three-dimensional model that involves apoptosis of the central acinar cells. Calmodulin kinase IID (CaMKIID) was selected as a candidate for the kinase required for Thr-457 phosphorylation from a gene chip analysis comparing genes up-regulated in MCF7 cells expressing wild type CEACAM1-SF compared with the T457A-mutated gene (Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). Up-regulation 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 co-workers (Maly, D. J., Allen, J. A., and Shokat, K. M. (2004) J. Am. Chem. Soc. 126, 9160-9161). 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.
Highlights
Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), a cell-cell adhesion molecule that induces lumen formation, requires phosphorylation on Thr457 for its function
Materials—Monoclonal antibodies anti-Calmodulin kinase IID (CaMKIID) and antiPKC␦ were from Abnova (Taipei, Taiwan); polyclonal antiPKC␦ was from Santa Cruz Biotechnology (Santa Cruz, CA); anti--actin was from Abcam (Cambridge, MA); anti-biotin was from Thermo Scientific (Lafayette, CO)
To make a positive identification of the bands tentatively identified for CaMKIID and casein kinase I (CKI), vector and wild type CEACAM1-SF-transfected MCF7 lysates were incubated with peptide and cross-linker; proteins were separated by SDS-gel electrophoresis and immunoblotted with antibodies to CaMKIID or CKI (Fig. 3C)
Summary
CEACAM1, a cell-cell adhesion molecule that induces lumen formation, requires phosphorylation on Thr457 for its function. 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 three-dimensional model that involves apoptosis of the central acinar cells. MCF7 cells were transfected with either wild type CEACAM1-SF or the T457A,S459A null mutant, and the mRNA levels were compared on cells grown in the three-dimensional model for 4 days when lumen formation is most active [9] In this analysis we identified several key proteins involved in the apoptotic process, namely calpain-9 and PKC-␦, and found that calmodulin kinase IID (CaMKIID) was elevated by 2.67-fold (log base 2) in the wild type versus the mutant transfectants. We conclude that CaMKIID plays an essential role in lumen formation in this model system and that Thr-457 in the short cytoplasmic domain isoform of CEACAM1 is likely a critical target of this kinase
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