Characterization of protein kinase C‐mediated phosphorylation of the short cytoplasmic domain isoform of C‐CAM

Edlund, Magnus; Wikström, Kristina; Toomik, Reet; Ek, Pia; Öbrink, Björn

doi:10.1016/s0014-5793(98)00222-1

Cited by 37 publications

(42 citation statements)

References 25 publications

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“…We further conclude that the direct interaction of CEACAM1-4S with actin is physiologically important, and that CEACAM1-4S is a major effector in the cytoskeletal rearrangements required for lumen formation in this model. CEACAM1-4S reveals the presence of Thr 457 and Ser 459 , residues that have been previously shown to be phosphorylated in murine CEACAM1 and predicted to be phosphorylated in human CEACAM1 (35). To demonstrate that these residues are phosphorylated in human CEACAM1, CEACAM1-4S-transfected MCF7 cells were grown in three-dimensional culture with or without 32 P i , cells were harvested and lysed, CEACAM1 was immunoprecipitated and proteins run on SDS gels, and the gels probed for 32 P by autoradiography or Western blotting with anti-phospho-Ser/Thr antibodies.…”

Section: Confocal Analysis Of Wild-type and Mutant Ceacam1-4s-actinmentioning

confidence: 88%

Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

Chen

Kirshner

Sherman

et al. 2007

Journal of Biological Chemistry

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CEACAM1-4S (carcinoembryonic antigen cell CEACAM14 (carcinoembryonic antigen cell adhesion molecule 1) is a type I membrane glycoprotein that mediates homotypic cell adhesion and belongs to the CEA gene family (1). It has been shown to play a role in bacterial and viral uptake (2-5), in insulin clearance (6), angiogenesis (7), NK and T-cell regulation (8 -11), and tumor suppression (12, 13). Despite its proposed function as a cell-cell adhesion molecule, CEACAM1 is often found on the lumenal surface of epithelial cells, suggesting the possibility that it also plays a role in cell polarization and lumen formation. In this respect, we have recently shown that CEACAM1 is expressed on the lumenal surface of normal mammary epithelial cells (14), and that in a three-dimensional model of mammary morphogenesis, blocking its expression with either an antisense gene or anti-CEACAM1 antibodies blocks lumen formation (15, 16), while breast cancer cells, which lack CEACAM1 and fail to form lumena in a three-dimensional culture, are restored to normal when transfected with the epithelial specific CEACAM1-4S isoform (16). CEACAM1 gene expression has been shown to be down-regulated early in colon cancer (12,17), and in a variety of cancer models, re-introduction of the CEACAM1 gene causes reversion of the malignant phenotype (18 -20). Although CEACAM1 is expressed in multiple isoforms with 1-4 Ig-like ectodomains and either long (72 amino acids) or short (12-14 amino acids) cytoplasmic domains, the short cytoplasmic domain isoforms predominate in epithelial cells. Because the cytoplasmic domain of this isoform is only 12-14 amino acids in length (the exact start of the domain is in doubt) it was originally thought not to play a role in signal transduction, but perhaps acted in a dominant negative fashion in the presence of the long cytoplasmic domain. However, we have shown that peptides or glutathione S-transferase fusion proteins from the short cytoplasmic domain bind directly to actin, tropomyosin, calmodulin, and more recently, to the annexin-2 p11 tetramer AIIt (21-23).In this study, we have used mutational analysis of the short cytoplasmic domain of CEACAM1 to reveal which residues are involved in actin interactions and lumen formation. The results of our studies show that mutants F454A and K456A greatly decrease or increase, respectively, actin interactions, whereas the F454A and the double mutant T457A,S459A greatly reduce lumen formation. Further analysis reveals that either Thr 457 or Ser 459 are phosphorylated during lumen formation. The studies that identified these key residues are the subject of this report. MATERIALS AND METHODS Construction of Baits for Yeast Two-hybrid ScreeningThe nucleotide and amino acid sequence for CEACAM1-4S is taken from NCBI entry number NM_001712, gi:4502404, the full-length coding sequence including the N-terminal signal * This work was supported by National Institutes of Health NCI Grant CA84202. The costs of publication of this article were defrayed in part by the payment of page ch...

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Section: Confocal Analysis Of Wild-type and Mutant Ceacam1-4s-actinmentioning

confidence: 88%

Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

Chen

Kirshner

Sherman

et al. 2007

Journal of Biological Chemistry

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“…Calmodulin inhibitors block the interaction of calmodulin with L-selectin and induce rapid L-selectin shedding upon treatment of neutrophils [50]. It is well established that the association of calmodulin with a number of ligands is regulated by PKC phosphorylation of the calmodulin-binding region [51][52][53]. We are currently examining whether the phosphorylation of L-selectin regulates calmodulin binding.…”

Section: Discussionmentioning

confidence: 99%

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils

Alexander

Kishimoto

Walcheck

2000

Journal of Leukocyte Biology

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The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced Lselectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP-and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/ CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce Lselectin shedding from neutrophils. J. Leukoc. Biol. 67: 415-422; 2000.

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“…We first considered posttranslational changes such as phosphorylation of Thr and Ser residues. Previous work by Obrink and co-workers have shown that Thr and Ser residues in rat CEACAM1-4S can be phosphorylated by protein kinase C isozymes (Edlund et al, 1998), and recent work in our lab have shown residues Thr457 and Ser459 in human CEACAM1-4S were transiently phosphorylated during glandular lumen formation in vitro (Chen et al, 2007). To test the hypothesis that phosphorylation of these residues was responsible for the differences seen between the two model systems, we generated mutants T457D, T457A, S459D, S459A, T457D þ S459D and T457A þ S459A.…”

Section: Ceacam1 Isoforms In Lumen Formationmentioning

confidence: 98%

Role of CEACAM1 isoforms in an in vivo model of mammary morphogenesis: mutational analysis of the cytoplasmic domain of CEACAM1-4S reveals key residues involved in lumen formation

Yokoyama

Nguyen

et al. 2007

Oncogene

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CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) is a type I transmembrane glycoprotein expressed in epithelial cells with three or four extracellular domains (ECDs) and either long or short cytoplasmic domain isoforms. We have previously shown that the four extracellular domains, short cytoplasmic domain isoform, CEACAM1-4S, plays an essential role in lumen formation in an in vitro model of mammary morphogenesis. In this study, we transfected MCF-7 cells with either the long or short cytoplasmic domain isoforms of CEACAM1, and grew the cells in humanized mammary mouse fat pads in NOD/SCID mice. In this in vivo model, only the long cytoplasmic domain isoform, CEACAM1-4L, formed glands with lumen. On the basis of other studies that revealed phosphorylation of key Thr and Ser residues in the short cytoplasmic domain, we introduced phosphorylation mimic (for example, Thr or Ser to Asp) or null (Thr or Ser to Ala) mutations into the cytoplasmic domain of CEACAM1-4S and tested them in the in vivo model. Mutation of either Thr or Ser to Asp or the double mutant Thr þ Ser to Asp, but not the null mutants, induced gland formation with a central lumen-containing apoptotic cells. Moreover, the phosphorylation mimic mutants of CEACAM1-4S induced downregulation of b1-integrin, overexpression of b2-integrin, inhibited phosphorylation of focal adhesion kinase (pTyr-397) and resulted in myofibroblast differentiation as characterized by expression of vimentin, a-smooth muscle actin and b2-integrin, as well as the production of abundant extracellular matrix.

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Characterization of protein kinase C‐mediated phosphorylation of the short cytoplasmic domain isoform of C‐CAM

Cited by 37 publications

References 25 publications

Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils

Role of CEACAM1 isoforms in an in vivo model of mammary morphogenesis: mutational analysis of the cytoplasmic domain of CEACAM1-4S reveals key residues involved in lumen formation

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