Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable 1 . This is thought to be due to the release of find-me signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages, and dendritic cells, leading to the prompt clearance of the dying cells 2 . However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or ectopic CD39 expression)Correspondence and requests for materials should be addressed to K.S.R. (ravi@virginia.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing interests.Author Contributions M.R.E. designed, performed and analyzed most of the experiments in this study with input from K.S.R. F.B.C. performed ATP quantitation experiments. P.T.C. helped with in vivo thymic apoptosis experiments. E.R.L. carried out HPLC analysis of supernatants. S.F.W. generated the CD39 expression plasmid and stable Jurkat cell lines. D.P. conducted phagocytosis experiments. A.K. and N.L. carried out the MS analysis and provided critical support in establishing the air-pouch model system. R.I.W. and J.J.L. carried out immunohistochemical detection of apoptotic cells in the thymus. M.O. and P.S. assisted with the BMDM generation and macrophage chemotaxis experiments. T.K.H. provided critical intellectual input in the preparation of the manuscript. K.S.R. provided overall coordination with respect to conception, design and supervision of the study. K.S.R. and M.R.E. wrote the manuscript with comments from co-authors. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 April 8. Most developing thymocytes (95%) undergo apoptosis; yet in steady-state only 1-2% are detectable as apoptotic 4,5 . It is hypothesized that dying thymocytes secrete soluble factors that attract resident phagocytes to promote prompt clearance 2,6 . To determine if apoptotic thymocytes release such factors, cell-free supernatants after apoptosis induction (by antiFas/CD95 crosslinking) were assessed for their ability to attract THP-1 monocytesor primary human monocytes in a transwell migration assay ( Figure 1a and Supplemental Figure S2). Apoptotic supernatants caused a 3-fold increase in monocyte migration compared to supernatants of live thymocytes. Such release of chemotactic factors was also seen with Jurkat cells (...

Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes1. The nucleotides ATP and UTP represent one class of find-me signals2, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 over-expression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes3. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.

Background: Pannexin 1 is activated by caspase cleavage of its C-terminal tail during apoptosis. Results: Cleavage removes a critical adjacent region to activate membrane-associated PANX1; activation requires dissociation of the C terminus from the pore. Conclusion: An intrinsic inhibitory interaction between the C terminus and the pore constrains PANX1 activity. Significance: PANX1 activation is caused by disruption of C-terminal-mediated inhibition.

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The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc⅐SHIP complex and regulation of SHIP function during T cell receptor signaling.The adapter protein Shc is a key regulator of intracellular signaling events that lead to such varied biological processes as neuronal differentiation, lymphocyte proliferation, and cellular transformation via polyoma virus middle T antigen (1-6). Shc mediates these effects, at least in part, through the activation of Ras proteins following stimulation of many receptors, including the receptors for growth factors (2, 7-10), antigens (11, 12), and cytokines (13-18), as well as G protein-coupled receptors (19). Shc contains an amino-terminal phosphotyrosine binding (PTB) 1 domain, a central collagen homology (CH) region, and a carboxyl-terminal Src homology 2 (SH2) domain but no apparent catalytic domain (7,20,21). In hematopoietic cells, Shc exists in two isoforms of 46 and 52 kDa. Upon activation of many receptors, Shc is tyrosine-phosphorylated and subsequently interacts with the SH2 domain of Grb2 (22,23). Grb2, in turn, interacts with the Ras GTP/GDP exchange factor, mSOS (24 -26). The complex of Shc⅐Grb2⅐mSOS becomes localized to the membrane through the association of Shc with the activated, tyrosine-phosphorylated receptors, where it leads to Ras activation (27). Shc interaction with activated receptors can occur either via the SH2 or the PTB domain. While both domains bind phosphotyrosine-containing sequences, their specificities of recognition are different and require residues either COOH-terminal (for SH2) or NH 2 -terminal (for PTB) to the Tyr(P) (28 -33). In hematopoietic cells, the Shc SH2 domain binds to components of the T cell receptor (TCR) (6, 11) and B cell receptor ...

The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosinebinding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.The adapter protein Shc is a critical regulator of downstream signaling events that lead to such diverse biological processes as neuronal differentiation (20,34,35,55), lymphocyte proliferation (31), cell survival mediated by cytokines and integrins (11,64), and cellular transformation via polyoma virus middle T antigen (3, 7). Shc mediates these effects, at least in part,