Identification of tubby and tubby-like protein 1 as eat-me signals by phage display

Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1^−/− mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.     

Unconventional secretion of tubby and tubby-like protein 1

Tubby-like proteins (Tulps) with no signal peptide have been characterized as cytoplasmic proteins with various intracellular functions, including binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. PI(4,5)P₂ has been implicated in unconventional secretion of fibroblast growth factor-2 without a signal peptide. Here, we show that all Tulps are expressed intracellularly and extracellularly. Tubby secretion is partially dependent on its PI(4,5)P₂-binding activity with an essential secretory signal in the N-terminus. Pathogenic mutation in Tubby mice has no impact on tubby extracellular trafficking. Moreover, unconventional secretion of tubby and Tulp1 is independent of endoplasmic reticulum-Golgi pathway. These data implicate that Tulps may function extracellularly as well.     

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.     

Galectin-3 is a new MerTK-specific eat-me signal

Phagocytosis of apoptotic cells and cellular debris is a critical process of maintaining tissue and immune homeostasis. Defects in the phagocytosis process cause autoimmunity and degenerative diseases. Phagocytosis ligands or "eat-me" signals control the initiation of the process by linking apoptotic cells to receptors on phagocyte surface and triggering signaling cascades for cargo engulfment. Eat-me signals are traditionally identified on a case-by-case basis with challenges, and the identification of their cognate receptors is equally daunting. Here, we identified galectin-3 (Gal-3) as a new MerTK ligand by an advanced dual functional cloning strategy, in which phagocytosis-based functional cloning is combined with receptor-based affinity cloning to directly identify receptor-specific eat-me signal. Gal-3 interaction with MerTK was independently verified by co-immunoprecipitation. Functional analyses showed that Gal-3 stimulated the phagocytosis of apoptotic cells and cellular debris by macrophages and retinal pigment epithelial cells with MerTK activation and autophosphorylation. The Gal-3-mediated phagocytosis was blocked by excessive soluble MerTK extracellular domain and lactose. These results suggest that Gal-3 is a legitimate MerTK-specific eat-me signal. The strategy of dual functional cloning with applicability to other phagocytic receptors will facilitate unbiased identification of their unknown ligands and improve our capacity for therapeutic modulation of phagocytic activity and innate immune response.     

The tubby family proteins

The tubby mouse shows a tripartite syndrome characterized by maturity-onset obesity, blindness and deafness. The causative gene Tub is the founding member of a family of related proteins present throughout the animal and plant kingdoms, each characterized by a signature carboxy-terminal tubby domain. This domain consists of a β barrel enclosing a central α helix and binds selectively to specific membrane phosphoinositides. The vertebrate family of tubby-like proteins (TULPs) includes the founding member TUB and the related TULPs, TULP1 to TULP4. Tulp1 is expressed in the retina and mutations in TULP1 cause retinitis pigmentosa in humans; Tulp3 is expressed ubiquitously in the mouse embryo and is important in sonic hedgehog (Shh)-mediated dorso-ventral patterning of the spinal cord. The amino terminus of these proteins is diverse and directs distinct functions. In the best-characterized example, the TULP3 amino terminus binds to the IFT-A complex, a complex important in intraflagellar transport in the primary cilia, through a short conserved domain. Thus, the tubby family proteins seem to serve as bipartite bridges through their phosphoinositide-binding tubby and unique amino-terminal functional domains, coordinating multiple signaling pathways, including ciliary G-protein-coupled receptor trafficking and Shh signaling. Molecular studies on this functionally diverse protein family are beginning to provide us with remarkable insights into the tubby-mouse syndrome and other related diseases.     

Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase

Diurnal phagocytosis of shed photoreceptor outer-segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing αv integrins upstream of tyrosine kinases and Rho GTPases. In this study, we tested the interdependence of the tyrosine kinases focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK) and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1, but not RhoA or Cdc42, during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires αvβ5 integrin and its ligand milk fat globule EGF factor-8 (MFG-E8) but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of αvβ5 toward MerTK by inhibiting FAK specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated αvβ5 integrin. Both pathways are independently activated and required for clearance phagocytosis.     

Efficient identification of tubby‐binding proteins by an improved system of T7 phage display

Mutation in the tubby gene causes adult‐onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby‐like protein 1 (Tulp1), whose C‐terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N‐terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N‐terminus (tubby‐N) as bait to identify unknown binding proteins with open‐reading‐frame (ORF) phage display. T7 phage display was engineered with three improvements: high‐quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait‐binding proteins in as fast as ～4–7 days. While phage display with conventional cDNA libraries identifies high percentage of out‐of‐frame unnatural short peptides, all 28 tubby‐N‐binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two‐hybrid assay and protein pull‐down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby‐specific binding protein. These data suggest that tubby‐N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly‐engineered ORF phage display is a powerful technology to identify unknown protein–protein interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Tubby proteins: the plot thickens

The tubby mouse, which shows late-onset obesity and neurosensory deficits, arises from a mutation in the Tub gene. Tub shares homology with the genes for tubby-like proteins Tulp1, Tulp2 and Tulp3. Ablation of Tub, Tulp1 or Tulp3 causes disease phenotypes that are indicative of their importance in nervous-system function and development. Despite this importance, the biochemical functions of tubby-like proteins are only now beginning to be understood. At present, data indicate that tubby-like proteins might function as heterotrimeric-G-protein-responsive intracellular signalling factors, although an array of data also implicates them in other processes.     

The first laminin G-like domain of protein S is essential for binding and activation of Tyro3 receptor and intracellular signalling

The homologous proteins Gas6 and protein S (ProS1) are both natural ligands for the TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases. ProS1 selectively activates Tyro3; however, the precise molecular interface of the ProS1-Tyro3 contact has not been characterised. We used a set of chimeric proteins in which each of the C-terminal laminin G-like (LG) domains of ProS1 were swapped with those of Gas6, as well as a set of ProS1 mutants with novel added glycosylations within LG1. Alongside wildtype ProS1, only the chimera containing ProS1 LG1 domain stimulated Tyro3 and Erk phosphorylation in human cancer cells, as determined by Western blot. In contrast, Gas6 and chimeras containing minimally the Gas6 LG1 domain stimulated Axl and Akt phosphorylation. We performed in silico homology modelling and molecular docking analysis to construct and evaluate structural models of both ProS1-Tyro3 and Gas6-Axl ligand-receptor interactions. These analyses revealed a contact between the ProS1 LG1 domain and the first immunoglobulin domain of Tyro3, which was similar to the Gas6-Axl interaction, and involved long-range electrostatic interactions that were further stabilised by hydrophobic and polar contacts. The mutant ProS1 proteins, which had added glycosylations within LG1 but which were all outside of the modelled contact region, all activated Tyro3 in cells with no hindrance. In conclusion, we show that the LG1 domain of ProS1 is necessary for activation of the Tyro3 receptor, involving protein-protein interaction interfaces that are homologous to those of the Gas6-Axl interaction.     

Endogenous Gas6 and Ca2+ -channel activation modulate phagocytosis by retinal pigment epithelium

Mutation or loss of MerTK as well as deficiency of vβ5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca²⁺-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca²⁺-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca²⁺-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca²⁺-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca²⁺-channels could be regulated downstream of both MerTK and vβ5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.     

Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

The C1b domain of protein kinase Cδ (PKCδ), a potent receptor for ligands such as diacylglycerol and phorbol esters, was synthesized by utilizing native chemical ligation. With this synthetic strategy, the domain was efficiently constructed and shown to have high affinity ligand binding and correct folding. The C1b domain has been utilized for the development of novel ligands for the control of phosphorylation by PKC family members. This strategy will pave the way for the efficient construction of C1b domains modified with fluorescent dyes, biotin, etc. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Structural basis for recruitment of the adaptor protein APS to the activated insulin receptor

The adaptor protein APS is a substrate of the insulin receptor and couples receptor activation with phosphorylation of Cbl to facilitate glucose uptake. The interaction with the activated insulin receptor is mediated by the Src homology 2 (SH2) domain of APS. Here, we present the crystal structure of the APS SH2 domain in complex with the phosphorylated tyrosine kinase domain of the insulin receptor. The structure reveals a novel dimeric configuration of the APS SH2 domain, wherein the C-terminal half of each protomer is structurally divergent from conventional, monomeric SH2 domains. The APS SH2 dimer engages two kinase molecules, with pTyr-1158 of the kinase activation loop bound in the canonical phosphotyrosine binding pocket of the SH2 domain and a second phosphotyrosine, pTyr-1162, coordinated by two lysine residues in beta strand D. This structure provides a molecular visualization of one of the initial downstream recruitment events following insulin activation of its dimeric receptor.     

Auto-oxidation and oligomerization of protein S on the apoptotic cell surface is required for Mer tyrosine kinase-mediated phagocytosis of apoptotic cells

Prompt phagocytosis of apoptotic cells prevents inflammatory and autoimmune responses to dying cells. We have previously shown that the blood anticoagulant factor protein S stimulates phagocytosis of apoptotic human B lymphoma cells by human monocyte-derived macrophages. In this study, we show that protein S must first undergo oxidative activation to stimulate phagocytosis. Binding of human protein S to apoptotic cells or to phosphatidylserine multilamellar vesicles promotes auto-oxidation of Cys residues in protein S, resulting in covalent, disulfide-linked dimers and oligomers that preferentially bind to and activate the human Mer tyrosine kinase (MerTK) receptor on the macrophages. The prophagocytic activity of protein S is eliminated when disulfide-mediated oligomerization is prevented, or when MerTK is blocked with neutralizing Abs. Protein S oligomerization is independent of phospholipid oxidation. The data suggest that membranes containing phosphatidylserine serve as a scaffold for protein S-protein S interactions and that the resulting auto-oxidation and oligomerization is required for the prophagocytic activity of protein S. In this way, apoptotic cells facilitate their own uptake by macrophages. The requirement for oxidative modification of protein S can explain why this abundant blood protein does not constitutively activate MerTK in circulating monocytes and tissue macrophages.     

Angiopoietin-1 and -2 coiled coil domains mediate distinct homo-oligomerization patterns, but fibrinogen-like domains mediate ligand activity.

Activity of endothelial Tie2 receptor tyrosine kinase is modulated by two naturally occurring, secreted ligands, angiopoietin-1 and -2, which have opposing effects on its phosphorylation. Receptor tyrosine kinase activation requires receptor dimerization/multimerization, which, for many receptors, is mediated by homo-oligomeric ligands binding to and bridging receptor molecules. We show here that angiopoietin-1 and -2 form distinct arrays of disulfide-linked homo-oligomeric complexes. Their mobilities on nonreducing gels suggest that angiopoietin-2 exists predominantly as a homodimer but also forms higher order multimers. In contrast, angiopoietin-1 forms some homotrimers, but predominantly exists in higher order multimers. These two structurally related, 60% homologous ligands are predominantly composed of an amino-terminal coiled coil domain and a carboxyl-terminal fibrinogen-like domain. We show that their distinct oligomerization patterns are determined by their coiled coil domains and, furthermore, that their coiled coil domains, but not their fibrinogen-like domains, are sufficient to mediate formation of disulfide-linked homo-oligomers. In contrast, the differential effects of these ligands on endothelial Tie2 phosphorylation is mediated by their fibrinogen-like domains. We conclude from these studies that the coiled coil and fibrinogen-like domains of the angiopoietins have distinct functions with the coiled coil domain mediating ligand homo-oligomerization and the fibrinogen-like domain mediating ligand activity.     

Structures of rhodopsin kinase in different ligand states reveal key elements involved in G protein-coupled receptor kinase activation

G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1.(Mg2+)2.ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.     

Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation

The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.     

Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions

Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.