Phosphorylation of CEACAM1 molecule by calmodulin kinase IID in a three-dimensional model of mammary gland lumen formation

J Biol Chem. 2014 Jan 31;289(5):2934-45. doi: 10.1074/jbc.M113.496992. Epub 2013 Dec 3.

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.

Keywords: Apoptosis; Breast Cancer; CaMKII; Calmodulin; Cell Adhesion; RNA Interference (RNAi).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenocarcinoma / metabolism
  • Adenocarcinoma / pathology
  • Amino Acid Sequence
  • Antigens, CD / genetics
  • Antigens, CD / metabolism*
  • Apoptosis / physiology
  • Benzylamines / pharmacology
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism*
  • Cell Adhesion Molecules / genetics
  • Cell Adhesion Molecules / metabolism*
  • Cell Culture Techniques / methods
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Female
  • Humans
  • MCF-7 Cells
  • Mammary Glands, Human / anatomy & histology
  • Mammary Glands, Human / cytology*
  • Molecular Sequence Data
  • Mutagenesis
  • Oligonucleotide Array Sequence Analysis
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • RNA, Small Interfering / genetics
  • Sulfonamides / pharmacology

Substances

  • Antigens, CD
  • Benzylamines
  • CD66 antigens
  • Cell Adhesion Molecules
  • Protein Kinase Inhibitors
  • RNA, Small Interfering
  • Sulfonamides
  • KN 93
  • W 7
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2