ITAM-like signalling for efficient phagocytosis : The paradigm of the granulocyte receptor CEACAM3

Abstract

Human CEACAM3 is a tailor-made receptor of the innate immune system to fight pathogens exploiting epithelial CEACAM-family members for colonisation and invasion of their host. Previous studies established CEACAM3 as the receptor facilitating rapid phagocytosis and elimination of N. gonorrhoeae by human granulocytes. The studies reported here set out to shed light on the evolution of this highly specialised receptor and the associated signalling machinery.CEACAM3 arose from exon shuffling after radiation of CEACAM1 approximately 24 million years ago in an ancestor of modern primates, bringing together the N-terminal domain of an epithelial CEACAM (CEACAM1, CEA or CEACAM6) targeted by pathogens with an intracellular signalling sequence most probably derived from an early CEACAM4 equipping it with a trigger of efficient phagocytosis. This signalling motif, that is closer to a canonical ITAM in CEACAM4, was further diversified during CEACAM3 evolution. Phagocytosis via CEACAM3 requires the activity of Src-family tyrosine kinases and activation of the small GTPase Rac, a regulator of the actin cytoskeleton. We could show, that phosphorylation of the tyrosines in the CEACAM3 ITAM-like sequence allows direct recruitment of the guanine nucleotide exchange-factor (GEF) Vav, which acts on Rac. Rac triggers lamellipodia formation by its effector, the WAVE-complex, a heteropentameric assembly providing interfaces for Rac and the adapter protein Nck via Sra1 and Nap1 respectively. We show, that CEACAM3 orchestrates activation as well as localisation of the actin-polymerisation machinery by recruitment of the WAVE-complex via Nck, which constitutively binds the WAVE-complex and is recruited to sites of CEACAM3 phosphorylation in a SH2-dependent manner. Further studies were undertaken to elucidate the role of additional kinases in the CEACAM3 signalling pathway. From similarities to immunoreceptor-signalling pathways the Tec and Syk family of tyrosine kinases were in the focus of those studies. While Tec kinases were found to be important in uptake and directly bind to pY230 of CEACAM3 during uptake, Syk associated with CEACAM3 wildtype only after uptake, but not close to the plasma membrane during the phagocytic process. This is in contrast to a CEACAM3/CD3ζ-ITAM chimera, where Syk co-localised with the chimera and bacteria in early uptake but not intracellular bacteria. Together, these studies provide novel insight into the protein-interaction network initiated by bacterial binding to CEACAM3 and help to explain the efficient and rapid phagocytosis mediated by this granulocyte receptor.