Pro-cathepsin D prevents aberrant protein aggregation dependent on endoplasmic reticulum protein CLN6.

We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates.


p.Asn77Lys homozygous CLN6 mutation in two unrelated Japanese patients with Kufs disease, an adult onset neuronal ceroid lipofuscinosis.

Gene analysis results of the first patient revealed a homozygous mutation c231C>G, p.Asn77Lys in exon 3 and a homozygous c.297+48 A>T mutation in intron 3 in the CLN6 gene. The Asn amino acid is perfectly conserved among species. In silico analysis showed that the mutation is predicted to be probably damaging. Moreover, the second patient with Kufs disease also had the same homozygous mutations. These data suggest that the missense mutation must be pathogenic. Furthermore, the patients had lived in the same district; therefore, they both potentially inherited the founder effect mutations.


CLN6's luminal tail-mediated functional interference between CLN6 mutants as a novel pathomechanism for the neuronal ceroid lipofuscinoses.

CLN6 (Ceroid Lipofuscinosis, Neuronal, 6) is a 311-amino acid protein spanning the endoplasmic reticulum membrane. Mutations in CLN6 are linked to CLN6 disease, a hereditary neurodegenerative disorder categorized into the neuronal ceroid lipofuscinoses. CLN6 disease is an autosomal recessive disorder and individuals affected with this disease have two identical (homozygous) or two distinct (compound heterozygous) CLN6 mutant alleles. Little has been known about CLN6's physiological roles and the disease mechanism. We recently found that CLN6 prevents protein aggregate formation, pointing to impaired CLN6's anti-aggregate activity as a cause for the disease. To comprehensively understand the pathomechanism, overall anti-aggregate activity derived from two different CLN6 mutants needs to be investigated, considering patients compound heterozygous for CLN6 alleles. We focused on mutant combinations involving the S132CfsX18 (132fsX) prematurely terminated protein, produced from the most frequent mutation in CLN6. The 132fsX mutant nullified anti-aggregate activity of the P299L CLN6 missense mutant but not of wild-type CLN6. Wild-type CLN6's resistance to the 132fsX mutant was abolished by replacement of amino acids 297-301, including Pro297 and Pro299, with five alanine residues. Given that removal of CLN6's C-terminal fifteen amino acids 297-311 (luminal tail) did not affect the resistance, we suggested that CLN6's luminal tail, when unleashed from Pro297/299-mediated conformational constraints, is improperly positioned by the 132fsX mutant, thereby blocking the induction of anti- aggregate activity. We here reveal a novel mechanism for dissipating CLN6 mutants' residual functions, providing an explanation for the compound heterozygosity-driven pathogenesis.


Implications of graded reductions in CLN6's anti-aggregate activity for the development of the neuronal ceroid lipofuscinoses.

CLN6, spanning the endoplasmic reticulum membrane, is a protein of unknown function. Mutations in the CLN6 gene are linked to an autosomal recessively inherited disorder termed CLN6 disease, classified as a form of the neuronal ceroid lipofuscinoses (NCL). The pathogenesis of CLN6 disease remains poorly understood due to a lack of information about physiological roles CLN6 plays. We previously demonstrated that CLN6 has the ability to prevent protein aggregate formation, and thus hypothesized that the abrogation of CLN6's anti-aggregate activity underlies the development of CLN6 disease. To test this hypothesis, we narrowed down the region vital for CLN6's anti-aggregate activity, and subsequently investigated if pathogenic mutations within the region attenuate CLN6's anti-aggregate activity toward four aggregation-prone αB-crystallin (αBC) mutants. None of the four αBC mutants was prevented from aggregating by the Arg106ProfsX truncated CLN6 mutant, the human counterpart of the nclf mutant identified in a naturally occurring mouse model of late infantile-onset CLN6 disease. In contrast, the Arg149Cys and the Arg149His CLN6 mutants, both associated with adult-onset CLN6 disease, blocked aggregation of two out of and all of the four αBC mutants, respectively, indicating that CLN6's anti-aggregate activity is differentially modulated according to the substitution pattern at the same amino acid position. Collectively, we here propose that the graded reduction in CLN6's anti-aggregate activity governs the clinical course of late infantile- and adult-onset NCL.

Biochemical and Biophysical Research Communications


Identification of CLN6 as a molecular entity of endoplasmic reticulum-driven anti-aggregate activity

B-crystallin (BC) is a small heat shock protein. Mutations in the BC gene are linked to -crystallinopathy, a hereditary myopathy histologically characterized by intracellular accumulation of protein aggregates. The disease-causing R120G BC mutant, harboring an arginine-to-glycine replacement at position 120, is an aggregate-prone protein. We previously showed that the R120G mutant's aggregation in HeLa cells was prevented by enforced expression of BC on the endoplasmic reticulum (ER). To elucidate the molecular nature of the preventive effect on the R120G mutant, we isolated proteins binding to ER-anchored BC (TMBC). The ER transmembrane CLN6 protein was identified as a TMBC's binder. CLN6 knockdown in HeLa cells attenuated TMBC's anti-aggregate activity against the R120G mutant. Conversely, CLN6 overexpression enhanced the activity, indicating that CLN6 operates as a downstream effector of TMBC. CLN6 physically interacted with the R120G mutant, and repressed its aggregation in HeLa cells even when TMBC was not co-expressed. Furthermore, CLN6's antagonizing effect on the R120G mutant was compromised upon treatment with a lysosomal inhibitor, suggesting CLN6 requires the intact autophagy-lysosome system to prevent the R120G mutant from aggregating. We hence conclude that CLN6 is not only a molecular entity of the anti-aggregate activity conferred by the ER manipulation using TMBC, but also serves as a potential target of therapeutic interventions.


Prevention of aberrant protein aggregation by anchoring the molecular chaperone B-crystallin to the endoplasmic reticulum.

The chaperone B-crystallin (BC) is a member of the small heat shock protein family and its point or truncated mutants cause the muscular disorder -crystallinopathy. The illness is histologically characterized by accumulation of protein aggregates in muscle cells. Expression of the myopathy-causing R120G mutant of BC, harboring an arginine-to-glycine mutation at position 120, results in aggregate formation. We demonstrated that tethering BC to the endoplasmic reticulum (ER) membrane represses the protein aggregation mediated by the R120G mutant. ER-anchored BC decreased the amount of the R120G mutant through autophagic proteolysis. In contrast, knockdown of ATG5, an E3 ligase essential for autophagy, in ER-anchored BC-transfected cells restored the quantity of the R120G mutant. In this context, aggregate formation was still suppressed, indicating that ER-anchored BC profoundly constrains aggregation competency of the R120G mutant separately from downregulating the abundance of the mutant. We have proposed that protein aggregation is prevented by manipulation of the ER microenvironment with BC, and have shed light on a novel aspect of the ER as a therapeutic target.


Contribution of calumin to embryogenesis through participation in the endoplasmic reticulum-associated degradation activity.

Calumin is an endoplasmic reticulum (ER)-transmembrane protein, and little is known about its physiological roles. Here we showed that calumin homozygous mutant embryos die at embryonic days (E) 10.5-11.5. At mid-gestation, calumin was expressed predominantly in the yolk sac. Apoptosis was enhanced in calumin homozygous mutant yolk sacs at E9.5, pointing to a possible link to the embryonic lethality. Calumin co-immunoprecipitated with ERAD components such as p97, BIP, derlin-1, derlin-2 and VIMP, suggesting its involvement in ERAD. Indeed, calumin knockdown in HEK 293 cells resulted in ERAD being less efficient, as demonstrated by attenuation in both degradations of a misfolded 1-antitrypsin variant and the ER-to-cytosol dislocation of cholera toxin A1 subunit. In calumin homozygous mutant yolk sac endoderm cells, ER stress-associated alterations were observed, including lipid droplet accumulation, fragmentation of the ER and dissociation of ribosomes from the ER. In this context, the ER-overload response, assumed to be cytoprotective, was also triggered in the mutant endoderm cells, but seemed to fully counteract the excessive ER stress generated due to defective ERAD. Taken together, our findings suggested that calumin serves to maintain the yolk sac integrity through participation in the ERAD activity, contributing to embryonic development.


Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics.

Bone remodeling is regulated by local factors that regulate bone-forming osteoblasts and boneresorbing osteoclasts, in addition to hormonal activity. Recent studies have shown that reactive oxygen species (ROS) act as an intracellular signal mediator for osteoclast differentiation. However the role of ROS on osteoblast differentiation is poorly understood. Here, we investigated the impact of ROS on osteoblastic differentiation of MC3T3-E1 cells. Osteogenic induction resulted in notable enhancement of mineralization and expression of osteogenic marker gene alkaline phosphatase, which were accompanied by an increase in ROS production. Additionally, we found that mitochondrial morphology dynamically changed from tubular reticulum to fragmented structures during the differentiation, suggesting that mitochondrial morphological transition is a novel osteoblast differentiation index. The antioxidant N-acetyl cysteine prevented not only ROS production but also mineralization and mitochondrial fragmentation. It is therefore suggested that the ROSdependent signaling pathways play a role in osteoblast differentiation accompanied by mitochondrial morphological transition.


Protective role of the endoplasmic reticulum protein mitsugumin23 against ultraviolet C-induced cell death.

The endoplasmic reticulum (ER) operates in adaptive responses to various stresses, dictating cell fate. Here we show that knockdown of the ER protein mitsugumin23 (MG23) enhances cell death induced by ultraviolet C (UVC), which causes DNA damage. The small heat shock protein B-crystallin (BC) is identified as a MG23 binding molecule and its knockdown facilitates death of UVC-exposed cells. Conversely, BC lowered UVC sensitivity when expressed as an ER-anchored form. Taken together, the results suggest that MG23 plays a protective role against UVC by accumulating BC in the close vicinity of the ER.


Role for B-cell adapter for PI3K (BCAP) as a signaling adapter linking Toll-like receptors (TLRs) to serine/threonine kinases PI3K/Akt.

Toll like receptors (TLRs) use Toll-IL-1 receptor (TIR) domain-containing adapters, such as myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adapter inducing IFN- (TRIF), to induce activation of transcription factors, including NF-B, MAP kinases, and IFN regulatory factors. TLR signaling also leads to activation of PI3K, but the molecular mechanism is not understood. Here we have discovered a unique role for B-cell adapter for PI3K (BCAP) in the TLR-signaling pathway. We find that BCAP has a functional N-terminal TIR homology domain and links TLR signaling to activation of PI3K. In addition, BCAP negatively regulates proinflammatory cytokine secretion upon TLR stimulation. In vivo, the absence of BCAP leads to exaggerated recruitment of inflammatory myeloid cells following infections and enhanced susceptibility to dextran sulfate sodium-induced colitis. Our results demonstrate that BCAP is a unique TIR domain-containing TLR signaling adapter crucial for linking TLRs to PI3K activation and regulating the inflammatory response.

Proceedings of the National Academy of Sciences of the United States of America


A Requirement for the p85 PI3K Adapter Protein BCAP in the Protection of Macrophages from Apoptosis Induced by Endoplasmic Reticulum Stress.

Macrophages are innate immune cells that play key roles in regulation of the immune response and in tissue injury and repair. In response to specific innate immune stimuli, macrophages may exhibit signs of endoplasmic reticulum (ER) stress and progress to apoptosis. Factors that regulate macrophage survival under these conditions are poorly understood. In this study, we identified B cell adapter protein (BCAP), a p85 PI3K-binding adapter protein, in promoting survival in response to the combined challenge of LPS and ER stress. BCAP was unique among nine PI3K adapter proteins in being induced >10-fold in response to LPS. LPS-stimulated macrophages incubated with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase inhibitor that induces ER stress, underwent caspase-3 activation and apoptosis. Macrophages from BCAP(-/-) mice exhibited increased apoptosis in response to these stimuli. BCAP-deficient macrophages demonstrated decreased activation of Akt, but not ERK, and, unlike BCAP-deficient B cells, expressed normal amounts of the NF-B subunits, c-Rel and RelA. Retroviral transduction of BCAP-deficient macrophages with wild-type BCAP, but not a Y4F BCAP mutant defective in binding the SH2 domain of p85 PI3K, reversed the proapoptotic phenotype observed in BCAP-deficient macrophages. We conclude that BCAP is a nonredundant PI3K adapter protein in macrophages that is required for maximal cell survival in response to ER stress. We suggest that as macrophages engage their pathogenic targets, innate immune receptors trigger increased expression of BCAP, which endows them with the capacity to withstand further challenges from ongoing cellular insults, such as ER stress.


Role of cell surface H+-ATP synthase on adipocyte differentiation.


Facilitation of DNA damage-induced apoptosis by endoplasmic reticulum protein mitsugumin23.

The endoplasmic reticulum (ER) emanates context-dependent signals, thereby mediating cellular response to a variety of stresses. However, the underlying molecular mechanisms have been enigmatic. To better understand the signaling capacity of the ER, we focused on roles played by mitsugumin23 (MG23), a protein residing predominantly in this organelle. Overexpression of MG23 in human embryonic kidney 293T cells specifically enhanced apoptosis triggered by etoposide, a DNA-damaging anti-cancer drug. Conversely, genetic deletion of MG23 reduced susceptibility of thymocytes to DNA damage-induced apoptosis, which was demonstrated by whole-body irradiation experiments. In this setting, induction of the tumor-suppressor gene p53 was attenuated in MG23-knockout thymocytes as compared with their wild-type counterparts, consistent with the elevated radioresistance. It is therefore suggested that MG23 is an essential component of ER-generated lethal signals provoked upon DNA damage, specifying cell fate under pathophysiological conditions.

Biochemical and Biophysical Research Communications


Essential role of the TRIC-B channel in Ca2+ handling of alveolar epithelial cells and in perinatal lung maturation.

TRIC channels function as monovalent cation-specific channels that mediate counter ion movements coupled with ryanodine receptor-mediated Ca(2+) release from intracellular stores in muscle cells. Mammalian tissues differentially contain two TRIC channel subtypes: TRIC-A is abundantly expressed in excitable cells, whereas TRIC-B is ubiquitously expressed throughout tissues. Here, we report the physiological role of TRIC-B channels in mouse perinatal development. TRIC-B-knockout neonates were cyanotic owing to respiratory failure and died shortly after birth. In the mutant neonates, the deflated lungs exhibited severe histological defects, and alveolar type II epithelial cells displayed ultrastructural abnormalities. The metabolic conversion of glycogen into phospholipids was severely interrupted in the mutant type II cells, and surfactant phospholipids secreted into the alveolar space were insufficient in the mutant neonates. Moreover, the mutant type II cells were compromised for Ca(2+) release mediated by inositol-trisphosphate receptors, despite Ca(2+) overloading in intracellular stores. Our results indicate that TRIC-B channels take an active part in Ca(2+) signalling to establish specialised functions in type II cells and are thus essential for perinatal lung maturation.


Mitsugumin 53-mediated maintenance of K+ currents in cardiac myocytes.

Mitsugumin 53 (MG53) is a muscle-specific RBCC/TRIM family member predominantly localized on small vesicles underneath the plasma membrane. Upon cell-surface lesion MG53 recruits the vesicles to the repair site in an oxidation-dependent manner and MG53-knockout mice develop progressive myopathy associated with defective membrane repair. In this report, we focus on MG53-knockout cardiomyocytes showing abnormal action potential profile and a reduced K+ current density. In cDNA expression experiments using cultured cells, KV2.1-mediated currents were remarkably increased by MG53 without affecting the total and cell-surface levels of channel expression. In imaging analysis MG53 seemed to facilitate the mobility of KV2.1-containing endocytic vesicles with acidic pH. However, similar effects on the current density and vesicular mobility were not observed in the putative dominant-negative form of MG53. Our data suggest that MG53 is involved in a constitutive cycle of certain cell-surface proteins between the plasma membrane and endosome-like vesicles in striated muscle, and also imply that the vesicular dynamics are essential for the quality control of KV2.1 in cardiomyocytes.


Enhanced NK-cell development and function in BCAP-deficient mice.

In B lymphocytes, the B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) facilitates signaling from the antigen receptor. Mice lacking BCAP have a predominantly immature pool of B cells with impaired immune function and increased susceptibility to apoptosis. Unexpectedly, we have found that natural killer (NK) cells from BCAP-deficient mice are more mature, more long-lived, more resistant to apoptosis, and exhibit enhanced functional activity compared with NK cells from wild-type mice. Surprisingly, these effects are evident despite a severe impairment of the immunoreceptor tyrosine-based activation motif-mediated Akt signaling pathway. The seemingly paradoxical phenotype reveals inherent differences in the signals controlling the final maturation of B cells and NK cells, which depend on positive and negative signals, respectively. Both enhanced interferon-gamma responses and augmented maturation of NK cells in BCAP-deficient mice are independent of available MHC class I ligands. Our data support a model in which blunting of BCAP-mediated activation signaling in developing NK cells promotes functionality, terminal maturation, and long-term survival.


Regulation of B-cell development by BCAP and CD19 through their binding to phosphoinositide 3-kinase.

Despite the importance of phosphoinositide 3-kinase (PI3K) in B-cell development, its activation mechanism still remains elusive. In this study, we show that deletion of both BCAP and CD19 leads to an almost complete block of BCR-mediated Akt activation and to severe defects in generation of immature and mature B cells. The YXXM motifs in BCAP and CD19 are crucial for regulating B-cell development in that mutation of these motifs abrogated their ability to induce BCR-mediated Akt activation as well as to promote B-cell development. Furthermore, the developmental defect in CD19(-/-)BCAP(-/-) B cells was partly relieved by introducing a constitutively active form of PI3K or PDK1. Together, our data suggest that BCAP and CD19 have complementary roles in BCR-mediated PI3K activation, thereby, at least in part, contributing to B-cell development.


Abnormal features in mutant cerebellar Purkinje cells lacking junctophilins.

Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca(2+)-mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase Cgamma in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice.


Augmentation of drug-induced cell death by ER protein BRI3BP.

To determine the contribution of the endoplasmic reticulum (ER) to cell fate decision, we focused on BRI3-binding protein (BRI3BP) residing in this organelle. BRI3BP, when overexpressed, augmented the apoptosis of human embryonic kidney 293T cells challenged with drugs including the anti-cancer agent etoposide. In contrast, the knockdown of BRI3BP reduced the drug-triggered apoptosis. BRI3BP overexpression enhanced both mitochondrial cytochrome c release and caspase-3 activity in etoposide-treated cells. In response to etoposide, the ER reorganized into irregularly shaped lamellae in mock-transfected cells, whereas in BRI3BP-overexpressing cells, such reorganization was not observed. These observations suggest that BRI3BP is involved in the structural dynamics of the ER and affects mitochondrial viability. Taken together, BRI3BP, widely expressed in animal cell types, seems to possess a pro-apoptotic property and can potentiate drug-induced apoptosis.


Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum.

We have identified a novel endoplasmic reticulum (ER)-resident protein, named "calumin", which is expressed in various tissues. This protein has a molecular mass of approximately 60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment, and a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of binding Ca2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and neonatal lethality, fluorometric Ca2+ imaging detected insufficient Ca2+ contents in intracellular stores and attenuated store-operated Ca2+ entry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin plays an essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision.


BANK negatively regulates Akt activation and subsequent B cell responses.

BANK is an adaptor protein that is highly expressed in B cells. To investigate its physiological role, we generated BANK-deficient mice. BANK-deficient mice displayed enhanced germinal center formation and IgM production in response to T-dependent antigens, whereas this phenotype was blocked in CD40-BANK double knockout mice. Involvement of BANK in CD40 signaling was further demonstrated by in vitro analysis. CD40-mediated proliferation and survival were significantly increased in BANK-deficient B cells, with enhanced Akt activation, whereas introduction of dominant-negative Akt into BANK-deficient B cells suppressed the augmented CD40-mediated responses. Together, our findings suggest that BANK attenuates CD40-mediated Akt activation, thereby preventing hyperactive B cell responses.


Contribution of BCAP to maintenance of mature B cells through c-Rel.

Mice deficient in the B cell adaptor for phosphoinositide 3-kinase (BCAP) have reduced numbers of mature B lymphocytes, which show defects in cell survival and proliferation. We found here that the NF-kappa B (Rel) pathway was impaired in BCAP-deficient mature B cells and that NF-kappa B target genes, indispensable for cell survival and division, were not induced in response to B cell receptor (BCR) stimulation. Among the NF-kappa B (Rel) family, expression of c-Rel was specifically reduced in BCAP-deficient B cells. Retrovirus-mediated reintroduction of c-Rel restored the pool size of immunoglobulin (Ig)M(lo)IgD(hi) mature B cells in the spleen as well as proliferative responses to BCR stimulation. These results indicate BCAP is essential in the maintenance of mature B cells through functional coupling with c-Rel.


T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein-GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70-containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.


Role of Grb2 in EGF-stimulated EGFR internalization.

Grb2 is an adaptor molecule that couples membrane receptors such as the epidermal growth factor receptor (EGFR) to intracellular signaling pathways. To gain insight into the trafficking pathways followed by these molecules after activation by EGF, we visualized Grb2 and EGFR fused to GFP spectral variants in single live cells. In nonstimulated cells, Grb2-YFP was primarily localized diffusely in the cytoplasm, whereas EGFR-CFP was found on the plasma membrane and in endocytic structures localized in the perinuclear area. Within 1 minute of EGF stimulation, Grb2 redistributed to the plasma membrane where it bound EGFR-CFP in an SH2 dependent manner. The plasma membrane then began to dynamically ruffle, and Grb2-YFP and EGFR-CFP were found to internalize together in large macropinocytic structures. These structures were morphologically distinct from conventional, clathrin-derived endosomes and did not label with transferrin, AP-2 or clathrin heavy chain. Evidence that these structures did not require clathrin for internalization came from experiments showing that expression of the C-terminus of AP-180, which inhibited transferrin uptake, had no effect on EGF-induced internalization of EGFR. YFP-tagged Grb2 containing an inhibitory mutation in either N- or C-SH3 domain redistributed to the plasma membrane upon EGF stimulation, but the macropinocytic structures containing Grb2-YFP and EGFR-CFP did not translocate inward and appeared to remain tethered to the plasma membrane. This suggested that the Grb2 SH3 domain was responsible for coupling the membranes containing EGFR with downstream effectors involved in internalization of these membranes. Transferrin uptake was unaffected in the presence of all of the SH3 domain mutants, consistent with the EGF-stimulated EGFR internalization pathway being clathrin-independent. These results demonstrate a role for Grb2 in events associated with a macropinocytic internalization pathway for EGFR in activated cells.


Essential immunoregulatory role for BCAP in B cell development and function.

BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell--independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca(2+) mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.


CAST, a novel CD3epsilon-binding protein transducing activation signal for interleukin-2 production in T cells.

Antigen recognition through T cell receptor (TCR)-CD3 complex transduces signals into T cells, which regulate activation, function, and differentiation of T cells. The TCR-CD3 complex is composed of two signaling modules represented by CD3zeta and CD3epsilon. Signaling through CD3zeta has been extensively analyzed, but that via CD3epsilon, which is also crucial in immature thymocyte development, is still not clearly understood. We isolated cDNA encoding a novel CD3epsilon-binding protein CAST. CAST specifically interacts in vivo and in vitro with CD3epsilon but not with CD3zeta or FcRgamma via a unique membrane-proximal region of CD3epsilon. CAST is composed of 512 amino acids including a single tyrosine and undergoes tyrosine phosphorylation upon TCR stimulation. Overexpression of two dominant-negative types of CAST, a minimum CD3epsilon-binding domain and a tyrosine-mutant, strongly suppressed NFAT activation and interleukin-2 production. These results demonstrate that CAST serves as a component of preformed TCR complex and transduces activation signals upon TCR stimulation and represents a new signaling pathway via the CD3epsilon-containing TCR signaling module.


Cold Spring Harbor Symposia on Quantitative Biology


ZAP-70 association with T cell receptor zeta (TCRzeta): fluorescence imaging of dynamic changes upon cellular stimulation.

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRzeta chain expressed as a chimeric protein (TTzeta and Tzetazeta), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tzetazeta fused to green fluorescent protein (ZAP-70 GFP and Tzetazeta-GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRzeta chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTzeta was indicated using mutant ZAP-70 GFPs and a truncated zeta chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tzetazeta- GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRzeta-ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.


Resistance of Fc receptor- deficient mice to fatal glomerulonephritis.

Immune complex-mediated inflammation is a common mechanism of various autoimmune diseases. Glomerulonephritis (GN) is one of these diseases, and the main mechanism of the induction of GN has been unclear. We examined the contribution of Fc receptors in the induction of nephrotoxic GN by establishing and analyzing mice deficient in the Fc receptor gamma chain (FcRgamma). Whereas all wild-type mice died from severe glomerulonephritis with hypernitremia by administration of anti-glomerular basement membrane (GBM) antibodies, all FcRgamma-deficient mice survived. Histologically, wild-type mice showed glomerular hypercellularity and thrombotic changes, whereas the renal tissue in FcRgamma-deficient mice was almost intact. Deposition of anti-GBM antibody as well as complement components in the GBM were equally observed in both wild-type and knockout mice. These results demonstrate that the triggering of this type of glomerulonephritis is completely dependent on FcR+ cells.


A shift from negative to positive selection of autoreactive T cells by the reduced level of TCR signal in TCR-transgenic CD3 zeta-deficient mice.

T cell selection is thought to be determined through the interaction between TCR and Ag/MHC. However, the contribution of the level of TCR signal to thymic selection remains unclear. To address this issue, we analyzed T cell selection of male Ag (HY)-specific TCR transgenic (HYTg) mice crossed with CD3 zeta-deficient (zeta KO) mice (HYTg/zeta KO), which have impaired signaling through TCR. In male HYTg/zeta KO mice, the number of thymocytes was comparable to that in normal mice, and almost all the peripheral T cells were HY specific, although these positively selected cells were anergic to male Ag. From these observations, the decrease in TCR signaling by CD3 zeta deficiency resulted in both the avoidance of negative selection and the acquisition of positive selection of autoreactive T cells in male HYTg/zeta KO mice. There was a shift of T cell selection from positive to no selection of HY-specific T cells in female HYTg/zeta KO mice also. Collectively, these findings suggest that the level of TCR signal directly regulates T cell selection; furthermore, the findings have integrated the models of T cell selection into a concept based on the quantity of TCR signal.


Specific interaction of topoisomerase II beta and the CD3 epsilon chain of the T cell receptor complex.

T cell antigen receptor (TCR)-CD3 complex is composed of six different subunits: TCR alpha and TCR beta and CD3 gamma, CD3 delta, CD3 epsilon, and CD3 eta. Antigen recognition signals are transduced from TCR to the cytoplasm through the cytoplasmic domain of the CD3 chains. To understand the downstream signal transduction pathways, we cloned genes encoding proteins capable of binding to CD3 epsilon with a probe of glutathione S-transferase fused to the cytoplasmic region of CD3 epsilon. One of these clones was found to encode topoisomerase II beta (topoII beta). The binding region of CD3 epsilon is located within the N-terminal 12 amino acids containing the motif of a basic amino acid cluster. A similar motif was found in the gamma chain of Fc receptors (FcR gamma) but not in the CD33 eta chain, and indeed, FcR gamma but not CD3 eta bound to topoII beta. The binding region of topoII beta was determined to be the C terminus. Since this region appears to be the regulatory region of the enzymatic activity, the binding of CD3 epsilon might affect the function of topoII beta. Although topoII beta is localized mainly in the nucleus and CD3E is a membrane protein, we demonstrated the presence of CD3 epsilon in the nuclear fraction of thymocytes, which increased upon T cell activation. The specific interaction in cells was evidenced by co-immunoprecipitation of topoII beta and CD3E from the nuclear fraction of T cells. The possible function of this interaction is discussed.


Induction of G1 arrest by down-regulation of cyclin D3 in T cell hybridomas.

The relationship between activation-induced growth inhibition and regulation of the cell cycle progression was investigated in T cell hybridomas by studying the function of the cell cycle-regulating genes such as G1 cyclins and their associated kinases. Activation of T cell hybridomas by anti-T cell receptor antibody induces growth arrest at G1 phase of the cell cycle and subsequently results in activation-driven cell death. Rapid reduction of both messenger RNA and protein level of the cyclin D3 is accompanied by growth arrest upon activation. Although the residual cyclin D3 protein forms a complex with cdk4 protein, cyclin D3-dependent kinase activity is severely impaired. Stable transfectants engineered to express cyclin D3 override the growth arrest upon activation. These results imply that the activation signal through T cell receptor induces the down-regulation of cyclin D3 expression and cyclin D3-dependent kinase activity, leading to growth arrest in G1 phase of the cell cycle in T cells.


Purification of glutathione S-transferase fusion proteins as a non-degraded form by using a protease-negative E. coli strain, AD202.


CTSD integrity in the endoplasmic reticulum is required for CLN6's anti-aggregate activity


Novel insight into the compound heterozygosity-driven CLN6 disease pathomechanism