Phosphatidylserine recognition by phagocytes: a view to a kill.

The redistribution of phosphatidylserine (PS) to the external surface of the plasma membrane is a key element of apoptotic cell recognition and is a molecular cue that dying cells should be engulfed. Phagocytes interact with PS on apoptotic cells through either the PS receptor or secreted bridging proteins called opsonins. The study of two secreted PS opsonins, MFG-E8 and Gas6 and their receptors alphavbeta5 (and alphavbeta3) integrin and Mer tyrosine kinase, respectively, have provided insights into the temporal and spatial aspects of Rac1 activation following the recognition and internalization of apoptotic cells. Disruption of PS opsonins and their signaling pathways often manifest conditions of inflammation and autoimmune disease. Here, we review recent studies involving PS opsonins, their receptors and their role in the phagocytosis of apoptotic cells.

Autophosphorylation docking site Tyr-867 in Mer receptor tyrosine kinase allows for dissociation of multiple signaling pathways for phagocytosis of apoptotic cells and down-modulation of lipopolysaccharide-inducible NF-kappaB transcriptional activation.

Efficient clearance of apoptotic cells is essential for tissue homeostasis, allowing for cellular turnover without inflammatory consequences. The Mer (Nyk and c-Eyk) receptor tyrosine kinase (Mertk) is involved in two aspects of apoptotic cell clearance by acting as a receptor for Gas6, a gamma-carboxylated phosphatidylserine-binding protein that bridges apoptotic and viable cells. First, Mertk acts in a bona fide engulfment pathway in concert with alphavbeta5 integrin by regulating cytoskeletal assemblages, and second, it acts as a negative regulator for inflammation by down-modulating pro-inflammatory signals mediated from bacterial lipopolysaccharide-Toll-like receptor 4 (TLR4) signaling, and hence recapitulating anti-inflammatory immune modulation by apoptotic cells. Here we describe Mertk post-receptor events that govern phagocytosis and cytoskeletal signaling are principally mediated by autophosphorylation site Tyr-867. Using the Mertk Y867F mutant and pharmacological inhibitors, we show that Tyr-867 is required for phosphatidylinositol 3-kinase and phospholipase Cgamma2 activation; their activation in turn elicits protein kinase C-dependent signals that act on the actin cytoskeleton. Although Mertk(Y867F) blocked the tyrosine phosphorylation of FAK on Tyr-861 and p130(cas) and also abrogated the phagocytosis of apoptotic cells, this mutant did not suppress lipopolysaccharide-inducible NF-kappaB transcription, nor was NF-kappaB activation dependent on the protein kinase C inhibitor, calphostin C. Finally, unlike the cytoskeletal events associated with Tyr-867 autophosphorylation, the trans-inhibition of NF-kappaB occurred in a postnuclear-dependent fashion independent of cytosolic IkappaB phosphorylation and p65/RelA sequestration. Taken together, these data suggest that Mertk has distinct and separable effects for phagocytosis and for resolving inflammation, providing a molecular rationale for how immune licensing and inflammation can be dissociated from phagocytosis in a single phagocytic receptor.

Characterization of the biochemical and biophysical properties of the phosphatidylserine receptor (PS-R) gene product.

The PS-R gene product was originally described as a cell surface receptor that interacts with externalized phosphatidylserine (PS) on apoptotic cells, but more recent studies have shown that it plays a critical role in organ development and terminal differentiation of many cell types during embryogenesis. Despite these important developmental functions, the biochemical and molecular properties of PS-R are poorly understood. Here we have used several approaches to show that PS-R undergoes processive post-translational protein cross-linking to form covalent multimers within the nuclear compartment. Although PS-R has a potential Glu-Glu (QQ) duet that is often targeted by transglutaminase TG-2, the oligomerization of PS-R was not effected by QQ-->AA mutation, or when PS-R gene product was expressed in TG-2 (-/-) fibroblasts. Pulse-chase experiments with (35) S-methionine indicates that the PS-R undergoes an initial proteolytic cleavage, followed by progressive multimerization of the monomeric subunits over time. In summary, we report here that PS-R is modified by an unusual post-translational modification, and we speculate that homomultimer of PS-R might be playing an important function as a scaffolding protein in the nucleus.

The presumptive phosphatidylserine receptor is dispensable for innate anti-inflammatory recognition and clearance of apoptotic cells.

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR(+/+), PSR(+/-), and PSR(-/-) fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells.

C-terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac-GEF.

Genetic studies in Caenorhabditis elegans identified an evolutionarily conserved CED-2 (CrkII), CED-5 (DOCK180), CED-12 (ELMO), CED-10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain-independent Rac guanine nucleotide exchange factor (Rac-GEF); but it is still unclear how CrkII functions in Rac-GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C-terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C-terminal SH3 domain of CrkII (CrkII-SH3-C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac-GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3-N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N-terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C-terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac-GEF.