The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

Lehrstuhl Zellbiologie, Universität Konstanz, Konstanz, Germany.
PLoS ONE (Impact Factor: 3.23). 03/2012; 7(3):e32808. DOI: 10.1371/journal.pone.0032808
Source: PubMed


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.
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.
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.

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    • "Previously, CEACAM3 has been shown to be a substrate of Src family PTKs expressed by human granulocytes (McCaw et al., 2003; Schmitter et al., 2004; Schmitter et al., 2007a). In vitro, the CEACAM3 ITAM-like motif is efficiently phosphorylated by active viral Src (v-Src) (Buntru et al., 2011; Pils et al., 2012). Therefore, we transfected 293 cells with constructs encoding GFP-tagged CEACAM3, CEACAM4, or the CEACAM3/4 chimera together with or without a v-Src expression plasmid. "
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    • "Due to its exceptional phagocytosis promoting properties, CEACAM3-initiated signaling has been studied in great detail (for review see [9]). In contrast to epithelial CEACAMs, CEACAM3-initiated uptake of bacteria critically relies on a cytoplasmic sequence motif and involves extensive actin cytoskeleton rearrangements orchestrated by the small GTPase Rac and its effector protein WAVE2 [8,62]. Importantly, CEACAM3-mediated phagocytosis is independent of sphingolipid- and cholesterol-rich membrane microdomains, as cholesterol chelators do not interfere with this process [61,63]. "
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    ABSTRACT: Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a group of immunoglobulin-related vertebrate glycoproteins. Several family members, including CEACAM1, CEA, and CEACAM6, are found on epithelial tissues throughout the human body. As they modulate diverse cellular functions, their signaling capacity is in the focus of current research. In this review we will summarize the knowledge about common signaling processes initiated by epithelial CEACAMs and suggest a model of signal transduction by CEACAM family members lacking significant cytoplasmic domains. As pathogenic and non-pathogenic bacteria exploit these receptors during mucosal colonization, we try to highlight the connection between CEACAMs, microbes, and cellular responses. Special emphasis in this context is placed on the functional interplay between CEACAMs and integrins that influences matrix adhesion of epithelial cells. The cooperation between these two receptor families provides an intriguing example of the fine tuning of cellular responses and their manipulation by specialized microorganisms.
    Cell Communication and Signaling 04/2014; 12(1):27. DOI:10.1186/1478-811X-12-27 · 3.38 Impact Factor
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    • "The phosphorylated cytoplasmic domain of CEACAM3 coordinates the local assembly of a signalling complex, which is responsible for the efficient internalization of bound bacteria [23]–[25]. By directly associating with the SH2 domains of the guanine nucleotide exchange factor (GEF) Vav and the adaptor molecule Nck, CEACAM3 recruits an upstream stimulator of the small GTPase Rac (the GEF Vav) and a downstream effector of GTP-loaded Rac (the Nck-associated WAVE-complex) to trigger massive actin rearrangements required for CEACAM3-mediated phagocytosis [26], [27]. The involvement of the CEACAM3 cytoplasmic domain and the strict requirement for dynamic actin rearrangements is clearly distinct from epithelial CEACAMs, which mediate endocytosis in the absence of a cytoplasmic domain. "
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    ABSTRACT: Several pathogenic bacteria utilize receptors of the CEACAM family to attach to human cells. Binding to different members of this receptor family can result in uptake of the bacteria. Uptake of Neisseria gonorrhoeae, a gram-negative human pathogen, via CEACAMs found on epithelial cells, such as CEACAM1, CEA or CEACAM6, differs mechanistically from phagocytosis mediated by CEACAM3, a CEACAM family member expressed selectively by human granulocytes. We find that CEACAM1- as well as CEACAM3-mediated bacterial internalization are accompanied by a rapid increase in phosphatidylinositol-3,4,5 phosphate (PI(3,4,5)P) at the site of bacterial entry. However, pharmacological inhibition of phosphatidylinositol-3' kinase (PI3K) selectively affects CEACAM1-mediated uptake of Neisseria gonorrhoeae. Accordingly, overexpression of the PI(3,4,5)P phosphatase SHIP diminishes and expression of a constitutive active PI3K increases CEACAM1-mediated internalization of gonococci, without influencing uptake by CEACAM3. Furthermore, bacterial uptake by GPI-linked members of the CEACAM family (CEA and CEACAM6) and CEACAM1-mediated internalization of N. meningitidis by endothelial cells require PI3K activity. Sensitivity of CEACAM1-mediated uptake toward PI3K inhibition is independent of receptor localization in cholesterol-rich membrane microdomains and does not require the cytoplasmic or the transmembrane domain of CEACAM1. However, PI3K inhibitor sensitivity requires the Ig(C2)-like domains of CEACAM1, which are also present in CEA and CEACAM6, but which are absent from CEACAM3. Accordingly, overexpression of CEACAM1 Ig(C2) domains blocks CEACAM1-mediated internalization. Our results provide novel mechanistic insight into CEACAM1-mediated endocytosis and suggest that epithelial CEACAMs associate in cis with other membrane receptor(s) via their extracellular domains to trigger bacterial uptake in a PI3K-dependent manner.
    PLoS ONE 06/2012; 7(6):e39908. DOI:10.1371/journal.pone.0039908 · 3.23 Impact Factor
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