Apoptotic cells are rapidly engulfed by phagocytes to prevent the release of potentially noxious or immunogenic intracellular materials from the dying cells, thereby preserving the integrity and ...function of the surrounding tissue. Phagocytes engulf apoptotic but not healthy cells, indicating that the apoptotic cells present a signal to the phagocytes, and the phagocytes recognize the signal using a specific receptor. Here, we report a factor that links apoptotic cells to phagocytes. We found that milk fat globule-EGF-factor 8 (MFG-E8), a secreted glycoprotein, was produced by thioglycollate-elicited macrophages. MFG-E8 specifically bound to apoptotic cells by recognizing aminophospholipids such as phosphatidylserine. MFG-E8, when engaged by phospholipids, bound to cells via its RGD (arginine-glycine-aspartate) motif-it bound particularly strongly to cells expressing αvβ3 integrin. The NIH3T3 cell transformants that expressed a high level of αvβ3 integrin were found to engulf apoptotic cells when MFG-E8 was added. MFG-E8 carrying a point mutation in the RGD motif behaved as a dominant-negative form, and inhibited the phagocytosis of apoptotic cells by peritoneal macrophages in vitro and in vivo. These results indicate that MFG-E8 secreted from activated macrophages binds to apoptotic cells, and brings them to phagocytes for engulfment.
Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in ...which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.
The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a ...Rho‐interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non‐hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho‐associated kinase (Rho‐kinase). Rho‐kinase has a catalytic domain in the N‐terminal portion, a coiled coil domain in the middle portion and a zinc finger‐like motif in the C‐terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho‐interacting interface. When COS7 cells were cotransfected with Rho‐kinase and activated RhoA, some Rho‐kinase was recruited to membranes. Thus it is likely that Rho‐kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho‐dependent signaling pathway.
Humanin is a secreted bioactive peptide that is protective in a variety of death models, including cell-based neuronal death models related to Alzheimer's disease (AD). To mediate the protective ...effect in AD-related death models, Humanin signals via a cell-surface receptor that is generally composed of three subunits: ciliary neurotrophic factor receptor α, WSX-1 and gp130 (heterotrimeric Humanin receptor; htHNR). However, the protective effect of Humanin via the htHNR is weak (EC50=1-10 μM); therefore, it is possible that another physiological agonist for this receptor exists in vivo. In the current study, calmodulin-like skin protein (CLSP), a calmodulin relative with an undefined function, was shown to be secreted and inhibit neuronal death via the htHNR with an EC50 of 10-100 pM. CLSP was highly expressed in the skin, and the concentration in circulating normal human blood was ~5 nM. When administered intraperitoneally in mice, recombinant CLSP was transported across the blood-cerebrospinal fluid (CSF)-barrier and its concentration in the CSF reaches 1/100 of its serum concentration at 1 h after injection. These findings suggest that CLSP is a physiological htHNR agonist.
Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion ...disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.
The small GTP‐binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism ...underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP‐bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP‐bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N‐terminus, followed by a coiled‐coil structure approximately 600 amino acids long, and a cysteine‐rich zinc finger‐like motif and a pleckstrin homology region in the C‐terminus. The N‐terminus region including a kinase domain and a part of coiled‐coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co‐expressed with RhoA in COS cells, p160 was co‐precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.
We report spectroscopic and photometric follow-up of the peculiar nova V5852 Sgr (discovered as OGLE-2015-NOVA-01), which exhibits a combination of features from different nova classes. The ...photometry shows a flat-topped light curve with quasi-periodic oscillations, then a smooth decline followed by two fainter recoveries in brightness. Spectroscopy with the Southern African Large Telescope shows first a classical nova with an Fe ii or Fe ii b spectral type. In the latter spectrum, broad emissions from helium, nitrogen and oxygen are prominent, and the iron has faded which could be an indication to the start of the nebular phase. The line widths suggest ejection velocities around 1000 km s−1. The nova is in the direction of the Galactic bulge and is heavily reddened by an uncertain amount. The V magnitude 16 days after maximum enables a distance to be estimated and this suggests that the nova may be in the extreme trailing stream of the Sagittarius dwarf spheroidal galaxy. If so it is the first nova to be detected from that, or from any dwarf spheroidal galaxy. Given the uncertainty of the method and the unusual light curve we cannot rule out the possibility that it is in the bulge or even the Galactic disk behind the bulge.
p47v‐crk (v‐Crk), a transforming gene product containing Src homology (SH)‐2 and ‐3 domains, induces an elevated level of tyrosine phosphorylation of several cellular proteins. Among these proteins, ...a 125‐135 kDa protein (p130) shows marked phosphorylation at tyrosines and tight association with v‐Crk, suggesting a direct signal mediator of v‐Crk. Here we report the molecular cloning of rat p130 by immunoaffinity purification. The p130 is a novel SH3‐containing signaling molecule with a cluster of multiple putative SH2‐binding motifs of v‐Crk. Immunochemical analyses revealed that p130 is highly phosphorylated at tyrosines during transformation by p60v‐src (v‐Src), as well as by v‐Crk, forming stable complexes with these oncoproteins. The p130 behaves as an extremely potent substrate of kinase activity included in the complexes and it is a major v‐Src‐associated substrate of the Src kinase by partial peptidase mapping. Subcellular fractionation demonstrated that the cytoplasmic p130 could move to the membrane upon tyrosine phosphorylation. The p130 (designated Cas for Crk‐associated substrate) is a common cellular target of phosphorylation signal via v‐Crk and v‐Src oncoproteins, and its unique structure indicates the possible role of p130Cas in assembling signals from multiple SH2‐containing molecules.
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