title: The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.
abstract: BACKGROUND
  CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake.
  
  
  RESULTS
  In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria.
  
  
  CONCLUSIONS
  Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.
authors.affiliation: Lehrstuhl Zellbiologie, Universität Konstanz, Konstanz, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pils
authors.forename: Kathrin
authors.initials: K
authors.lastname: Kopp
authors.forename: Lisa
authors.initials: L
authors.lastname: Peterson
authors.forename: Julia
authors.initials: J
authors.lastname: Delgado Tascón
authors.forename: Naja J
authors.initials: NJ
authors.lastname: Nyffenegger-Jann
authors.forename: Christof R
authors.initials: CR
authors.lastname: Hauck
mesh_terms: D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000378:metabolism*; D002478:Cells, Cultured; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D009344:Neisseria gonorrhoeae / Q000254:growth & development / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011554:Pseudopodia; D034741:RNA, Small Interfering / Q000235:genetics; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C075729:CEACAM3 protein, human; C082869:CRKL protein; D002272:Carcinoembryonic Antigen; C078332:Nck protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C092947:SLP-76 signal Transducing adaptor proteins; C114864:WASF1 protein, human; D051300:Wiskott-Aldrich Syndrome Protein Family