Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor.

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.

Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex.

Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer. We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF complex. We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA. Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling. Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1. The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair.

Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat.

We previously found hydroperoxide-responsive proteins (HPRPs), which are comprised of peroxiredoxin I (Prx I), Prx II, Prx III, Prx VI, HSP27, G3PDH and two unidentified proteins (HPRP-2' and HPRP-5'), in human umbilical vein endothelial cells. It was demonstrated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) that most HPRPs are converted into variants with lower pI upon exposure to hydroperoxides. In this study, we examined the HPRP response on 2D gels upon exposure of human endothelial cells (ECV304) to paraquat (PQ2+), which generates reactive oxygen species (ROS) within cells. PQ2+ exerted cytotoxic effects in a dose-(10 microM-10 mM) and time-(24-168 h) dependent manner. Two-dimensional PAGE analysis revealed that HPRP-2', and oxidized forms of Prx I, Prx II and Prx III were clearly increased upon exposure of cells to sublethal levels of PQ2+. Microsequence analysis revealed that both HPRP-2 and -2' were identical with human DJ-1. Moreover immunoblot analysis confirmed the increase of oxidized forms of Prx II, Prx III and DJ-1 in response to sublethal levels of PQ2+. PQ2+ treatment failed to increase fluorescence intensity derived from DCF, which is believed to be an indicator for intracellular levels of hydroperoxide. Although pentachlorophenol (PCP), an uncoupler of the mitochondrial respiratory chain, clearly elevated the fluorescence, PCP had no effect on HPRP response. These observations indicated that DCF-derived fluorescence is not correlated with HPRP response. We consider that the response of Prxs and DJ-1 on 2D gels could reflect endogenous production of ROS in PQ(2+)-treated cells, and might be a sensitive indicator of oxidative stress status.

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration.

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.

Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins.

The processes governing chromatin remodeling and assembly, which occur prior to and/or after transcription and replication, are not completely understood. To understand the mechanisms of transcription and replication from chromatin templates, we have established in vitro replication and transcription systems using adenovirus (Ad) DNA complexed with viral basic core proteins, called Ad core, as a template. Using this system, we have previously identified, from HeLa cells, template activating factor-I as a stimulatory factor for the Ad core DNA replication. Here, using this system as a tool, we identified and purified a novel template activating factor activity that consists of two acidic polypeptides whose apparent molecular masses are 38 kDa and 37 kDa. These two polypeptides correspond to two splicing variants of nucleolar phosphoprotein, nucleophosmin/B23. Recombinant B23 proteins stimulate the Ad core DNA replication, and the acidic regions of B23 proteins are important for its activity. In addition, B23 proteins directly bind to core histones and transfer them to naked DNA. Furthermore, chromatin components such as histones and topoisomerase II are co-immunoprecipitated with B23 from cell extracts. These observations lead to a hypothesis that nucleophosmin/B23 is involved in structural changes of chromatin, thereby regulating transcription and replication within the ribosomal DNA region or maintaining the nucleolar structure.

Variants of peroxiredoxins expression in response to hydroperoxide stress.

We examined patterns of the proteins that were expressed in human umbilical vein endothelial cells (HUVEC) in response to oxidative stress by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). When HUVEC were exposed to H2O2 at 100 microM for 60 min, the intensities of eight spots increased and those of eight spots decreased on 2D gels, as compared with control gels, after staining with silver. These changes were also observed after exposure of cells to hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide, but not after exposure to other reagents that induce oxidative stress such as S-alkylating compounds, nitric oxide, and salts of heavy metals. Therefore, these proteins were designated hydroperoxide responsive proteins (HPRPs). Microsequencing analysis revealed that these HPRPs corresponded to at least six pairs of proteins. Of these, four pairs of HPRPs were thioredoxin peroxidase I (TPx I), TPx II, TPx III, and the product of human ORF06, all of which belong to the peroxiredoxin (Prx) family and all of which are involved in the elimination of hydroperoxides. The other two pairs corresponded to heat shock protein 27 (HSP27) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH), respectively. The variants that appeared in response to hydroperoxides had molecular masses similar to the respective native forms, but their pI values were lower by 0.2-0.3 pH units than those of the corresponding native proteins. These variants were detected on 2D gels after cells had been exposed to hydroperoxides in the presence of an inhibitor of protein synthesis. All variants were generated within 30 min of exposure to 100 microM H2O2. The variants of TPx I and TPx II appeared within 2 min of the addition of H2O2 to the culture medium. The HPRPs returned to their respective native forms after the removal of stress. Our results indicated that at least six proteins were structurally modified in response to hydroperoxides. Analysis by 2D-PAGE of 32P-labeled proteins revealed that the variant of HSP27 was its phosphorylated form while the other HPRPs were not modified by phosphorylation. Taken together, the results suggest that 2D-PAGE can reveal initial responses to hydroperoxide stress at the level of protein modification. Moreover, it is possible that the variants of four types of Prx might reflect intermediate states in the process of hydroperoxide elimination.

An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization.

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein beta-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.

Regulation of Pim-1 by Hsp90.

The protooncogene Pim-1 encodes serine/threonine protein kinases that are involved in cytokine-mediated cell proliferation and in lymphoma- and leukemogenesis. It is largely unknown how Pim-1 executes its biological effects. Here we show that Pim-1 physically interacts with heat shock protein 90 alpha and beta (Hsp90alpha and beta). The Hsp90-specific inhibitor geldanamycin (GA) induced a rapid degradation of Pim-1 and reduced its kinase activity. The expression of Hsp90alpha was regulated by a signal from the cytokine receptor gp130, as is Pim-1's expression. These results indicate that Hsp90 is coordinately regulated with Pim-1 and is involved in the stabilization and function of Pim-1.

Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase.

In vertebrate cells, the nuclear entry of Cdc2-cyclin B1 (MPF) during prophase is thought to be essential for the induction and coordination of M-phase events. Phosphorylation of cyclin B1 is central to its nuclear translocation, but the kinases that are responsible remain unknown. Here we have purified a protein kinase from Xenopus M-phase extracts that phosphorylates a crucial serine residue (S147) in the middle of the nuclear export signal sequence of cyclin B1. We have identified this kinase as Plx1 (ref. 16), a Xenopus homologue of Polo-like kinase (Plk)-1. During cell-cycle progression in HeLa cells, a change in the kinase activity of endogenous Plk1 toward S147 and/or S133 correlates with a kinase activity in the cell extracts. An anti-Plk1 antibody depletes the M-phase extracts of the kinase activity toward S147 and/or S133. An anti-phospho-S147 antibody reacts specifically with cyclin B1 only during G2/M phase. A mutant cyclin B1 in which S133 and S147 are replaced by alanines remains in the cytoplasm, whereas wild-type cyclin B1 accumulates in the nucleus during prophase. Co-expression of constitutively active Plk1 stimulates nuclear entry of cyclin B1. Our results indicate that Plk1 may be involved in targeting MPF to the nucleus during prophase.

Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis.

Previous biochemical data identified a host cell fraction, designated RAF-2, which stimulated influenza virus RNA synthesis. A 48-kDa polypeptide (RAF-2p48), a cellular splicing factor belonging to the DEAD-box family of RNA-dependent ATPases previously designated BAT1 (also UAP56), has now been identified as essential for RAF-2 stimulatory activity. Additionally, RAF-2p48 was independently identified as an influenza virus nucleoprotein (NP)-interacting protein, NPI-5, in a yeast two-hybrid screen of a mammalian cDNA library. In vitro, RAF-2p48 interacted with free NP but not with NP bound to RNA, and the RAF-2p48-NP complex was dissociated following addition of free RNA. Furthermore, RAF-2p48 facilitated formation of the NP-RNA complexes that likely serve as templates for the viral RNA polymerase. RAF-2p48 was shown, in both in vitro binding assays and the yeast two-hybrid system, to bind to the amino-terminal region of NP, a domain essential for RNA binding. Together, these observations suggest that RAF-2p48 facilitates NP-RNA interaction, thus leading to enhanced influenza virus RNA synthesis.

Pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of alpha-ketoisocaproate for isoamyl alcohol formation in Saccharomyces cerevisiae.

Isoamyl alcohol is an important flavor component of yeast-fermented alcoholic beverages. To identify the enzyme and gene involved in the decarboxylation of alpha-ketoisocaproate (alpha-KIC) for isoamyl alcohol formation, the enzyme was partially purified and analyzed by mass spectrometry. The pyruvate decarboxylase encoded by the PDC1 gene was considered a likely candidate enzyme. Genetic analysis showed that the activity of alpha-KIC decarboxylase and production of isoamyl alcohol partially decreased in a pdc1 null mutant and increased in a transformant with a multi-copy plasmid carrying the PDC1 gene. These results indicate that pyruvate decarboxylase encoded by the PDC1 gene contributes, at least partially, to the decarboxylation of alpha-KIC for isoamyl alcohol formation.

Elongation factor-1 alpha is a novel substrate of rho-associated kinase.

Rho-associated kinase (Rho-kinase), which is activated by the Rho small GTPase, phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase, myosin light chain (MLC), the ERM family proteins, and adducin, thereby regulating the formation of stress fibers, focal adhesions, microvillus formation, and cell motility. Here, to further understand the role of Rho-kinase in the regulation of the numerous cellular processes by Rho, we purified a novel substrate of Rho-kinase having a molecular mass of 48 kDa (p48) from a rat liver cytosol extract. Mass spectral analysis revealed p48 to be elongation factor-1 alpha (EF-1 alpha), which is known as an actin-binding protein besides a cofactor of polypeptide elongation. Rho-kinase directly phosphorylated recombinant EF-1alpha in vitro. A high- speed cosedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the binding activity of EF-1 alpha to filamentous actin (F-actin). A low-speed sedimentation assay revealed that phosphorylation of EF-1 alpha by Rho-kinase decreased the F-actin-bundling activity. In addition, EF-1 alpha bound to MBS of myosin phosphatase, suggesting that both Rho-kinase and myosin phosphatase regulate the phosphorylation state of EF-1 alpha downstream of Rho as other substrates of Rho-kinase, i.e., MLC, adducin, and the ERM family. These results suggest that the Rho/Rho-kinase pathway regulates the organization of actin cytoskeleton via the phosphorylation of EF-1 alpha.

Novel responses of ZRF, a variant of human MTF-1, to in vivo treatment with heavy metals.

Heavy metal-dependent transcriptional activation of metallothionein (MT) genes is mediated by multiple enhancer sequences, metal responsive element (MRE), located in the upstream region of the genes. Previously, we have reported purification of a zinc-dependent MRE-binding protein, zinc regulatory factor (ZRF), from HeLa cells, and have pointed to the close relationship between ZRF and mouse MRE-binding transcription factor-1 (MTF-1) according to the analysis of partial amino acid sequences. By means of cDNA cloning and the product analyses, we show that ZRF is a variant of human MTF-1 (hMTF-1), which carries a single amino acid exchange in the zinc finger domain. Accordingly, ZRF is renamed hMTF-1b. Expression of hMTF-1b in HeLa cells is constitutive at both mRNA and protein levels, and is unaffected by treatment with cadmium (Cd). On the other hand, when cells were fractionated into nuclear extract and cytosol, a large part of the hMTF-1b was recovered in the cytosol fraction. A significant increase in the amount of nuclear hMTF-1b occurs when cells are treated with various heavy metals, including Cd, Zn, Cu and Ag, which is associated with concomitant decrease in the amount recovered in the cytosol fraction. Since immunocytochemical analysis revealed that intracellular distribution of hMTF-1b is restricted to the nucleus irrespective of the heavy metal treatment, such an increment in the nuclear extracts apparently results from promotion of nuclear retention of hMTF-1b by the heavy metal treatment. Analysis by native gel electrophoresis shows that the mobility of hMTF-1b significantly changes in association with Cd treatment, raising the possibility that a conformational change of hMTF-1b occurs in response to treatment with heavy metals in vivo.

Nitric oxide inactivates glyoxalase I in cooperation with glutathione.

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.

Zeta-crystallin catalyzes the reductive activation of 2,4,6-trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts.

Exposure to 2,4,6-trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role. From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species. The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS-PAGE. Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to zeta-crystallin. Superoxide anions were formed during reduction of TNT by zeta-crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens. Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.

Identification of a novel beta-catenin-interacting protein.

Cadherin is a well-known cell-cell adhesion molecule, and it binds to beta-catenin, which in turn binds to alpha-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell-cell adhesion. Here we purified two novel beta-catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)-beta-catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds beta-catenin and KIAA0313 in the yeast two-hybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456-461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38-44, 1999). beta-Catenin was coimmunoprecipitated with KIAA0313 in Madin-Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and beta-catenin were partly colocalized at sites of cell-cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with beta-catenin through KIAA0705 in vivo at sites of cell-cell contact.

A cysteine protease from maize isolated in a complex with cystatin.

We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize [Yamada et al. (1998) Plant Cell Physiol. 39: 106]. Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa). This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants. Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%). The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex. The protease expressed in E. coli itself was active but not latent, probably because it was not bound to cystatin. It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin. The mRNA of protease was expressed in various tissues except for seeds.

Phosphorylation of collapsin response mediator protein-2 by Rho-kinase. Evidence for two separate signaling pathways for growth cone collapse.

We previously identified Rho-associated protein kinase (Rho-kinase) as a specific effector of Rho. In this study, we identified collapsin response mediator protein-2 (CRMP-2), as a novel Rho-kinase substrate in the brain. CRMP-2 is a neuronal protein whose expression is up-regulated during development. Rho-kinase phosphorylated CRMP-2 at Thr-555 in vitro. We produced an antibody that specifically recognizes CRMP-2 phosphorylated at Thr-555. Using this antibody, we found that Rho-kinase phosphorylated CRMP-2 downstream of Rho in COS7 cells. Phosphorylation of CRMP-2 was observed in chick dorsal root ganglion neurons during lysophosphatidic acid (LPA)-induced growth cone collapse, whereas the phosphorylation was not detected during semaphorin-3A-induced growth cone collapse. Both LPA-induced CRMP-2 phosphorylation and LPA-induced growth cone collapse were inhibited by Rho-kinase inhibitor HA1077 or Y-32885. LPA-induced growth cone collapse was also blocked by a dominant negative form of Rho-kinase. On the other hand, semaphorin-3A-induced growth cone collapse was not inhibited by a dominant negative form of Rho-kinase. Furthermore, overexpression of a mutant CRMP-2 in which Thr-555 was replaced by Ala significantly inhibited LPA-induced growth cone collapse. These results demonstrate the existence of Rho-kinase-dependent and -independent pathways for growth cone collapse and suggest that CRMP-2 phosphorylation by Rho-kinase is involved in the former pathway.

A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster.

Caspase-activated DNase (CAD) is an enzyme that cleaves chromosomal DNA in apoptotic cells. Here, we identified a DNase in Drosophila Schneider cells that can be activated by caspase 3, and purified it as a complex of two subunits (p32 and p20). Using primers based on the amino acid sequence of the purified proteins, a cDNA coding for Drosophila CAD (dCAD) was cloned. The polypeptide encoded by the cDNA contained 450 amino acids with a calculated M(r) of 52,057, and showed significant homology with human and mouse CAD (22% identity). Mammalian CADs carry a nuclear localization signal at the C terminus. In contrast, dCAD lacked the corresponding sequence, and the purified dCAD did not cause DNA fragmentation in nuclei in a cell-free system. When dCAD was co-expressed in COS cells with Drosophila inhibitor of CAD (dICAD), a 52-kDa dCAD was produced as a heterotetrameric complex with dICAD. When the complex was treated with human caspase 3 or Drosophila caspase (drICE), the dICAD was cleaved, and released from dCAD. In addition, dCAD was also cleaved by these caspases, and behaved as a (p32)(2)(p20)(2) complex in gel filtration. When a Drosophila neuronal cell line was induced to apoptosis by treatment with a kinase inhibitor, both dCAD and dICAD were cleaved. These results indicated that unlike mammalian CAD, Drosophila CAD must be cleaved by caspases to be activated.