The myelin sheath is an essential structure for the rapid transmission of electrical impulses through axons, and peripheral myelination is a well-programmed postnatal process of Schwann cells (SCs), the myelin-forming peripheral glia. SCs transdifferentiate into demyelinating SCs (DSCs) to remove the myelin sheath during Wallerian degeneration after axonal injury and demyelinating neuropathies, and macrophages are responsible for the degradation of myelin under both conditions. In this study, the mechanism by which DSCs acquire the ability of myelin exocytosis was investigated. Using serial ultrastructural evaluation, we found that autophagy-related gene 7-dependent formation of a "secretory phagophore (SP) " and tubular phagophore was necessary for exocytosis of large myelin chambers by DSCs. DSCs seemed to utilize myelin membranes for SP formation and employed p62/sequestosome-1 (p62) as an autophagy receptor for myelin excretion. In addition, the acquisition of the myelin exocytosis ability of DSCs was associated with the decrease in canonical autolysosomal flux and was demonstrated by p62 secretion. Finally, this SC demyelination mechanism appeared to also function in inflammatory demyelinating neuropathies. Our findings show a novel autophagy-mediated myelin clearance mechanism by DSCs in response to nerve damage.

Pattern-recognition receptors including Toll-like receptors (TLRs) recognize invading pathogens and trigger an immune response in mammals. Here we show that mammalian ste20-like kinase 1/serine/threonine kinase 4 (MST1/STK4) functions as a negative regulator of lipopolysaccharide (LPS)-induced activation of the TLR4-NF-κB signaling pathway associated with inflammation. Myeloid-specific genetic ablation of MST1/STK4 increased the susceptibility of mice to LPS-induced septic shock. Ablation of MST1/STK4 also enhanced NF-κB activation triggered by LPS in bone marrow-derived macrophages (BMDMs), leading to increased production of proinflammatory cytokines by these cells. Furthermore, MST1/STK4 inhibited TRAF6 autoubiquitination as well as TRAF6-mediated downstream signaling induced by LPS. In addition, we found that TRAF6 mediates the LPS-induced activation of MST1/STK4 by catalyzing its ubiquitination, resulting in negative feedback regulation by MST1/STK4 of the LPS-induced pathway leading to cytokine production in macrophages. Together, our findings suggest that MST1/STK4 functions as a negative modulator of the LPS-induced NF-κB signaling pathway during macrophage activation.

Background. Sasa quelpaertensis Nakai extract (SQE) or dwarf bamboo has been extensively investigated for its antioxidant and anti-inflammatory effects; however, no previous study assessed its effect as an antidepressant agent. Therefore, this study was designed to examine the effect of oral SQE administration in ameliorating menopausal depressive symptoms and to evaluate its mechanisms in ovariectomized rats with repeated stress. Methods. All experimental groups except normal group underwent ovariectomy and then immobilization for 14 consecutive days. During these 2 weeks, two rat groups received SQE (100 and 300 mg/kg orally) and their cutaneous body temperature was measured. The tail suspension test (TST) and forced swim test (FST) were performed in order to evaluate depression-like behavior. Additionally, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were carried out to evaluate the central monoaminergic neurotransmitter levels and activity. Results. Oral SQE (100 mg/kg) administration had reduced immobility time in TST and FST. Additionally, the SQE 100 and 300 mg/kg administration had decreased the cutaneous body temperature in the rats compared to those without treatment. In ELISA analysis, the SQE 100 group expressed elevated levels of serotonin and dopamine in the hypothalamus, prefrontal cortex, and hippocampus. Antityrosine hydroxylase (anti-TH) antibodies showed a tremendous increase in the density of TH positive cells in the locus coeruleus (LC) region of the SQE 100 group. Likewise, the SQE 100 elevated the number of tryptophan hydroxylase (TPH) and protein kinase C (PKC) immunoreactive cell counts and density in the hypothalamic region. Conclusion. These results suggested that the oral SQE administration induced the antidepressant-like effect in the ovariectomized rats with repeated stress via upregulating the levels of serotonin and dopamine through enhancing the expression of TH, TPH, and PKC in many brain areas.

Sequestosome 1 (SQSTM1, p62), a ubiquitin binding protein, plays a role in cell signaling, oxidative stress, and autophagy. However, its functional role in inflammatory signaling is controversial. Recent studies have shown that p62 is negatively implicated in inflammatory responses. But, the precise molecular mechanisms by which p62 regulates inflammatory responses remain unclear. In this study, we report on a new regulatory role for p62 in TLR4-mediated signaling. p62 overexpression led to the suppression of NF-κB activation and the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β in response to TLR4 stimulation. In contrast, p62 -/- mouse embryonic fibroblast (MEF) cells exhibited marked enhancement of NF-κB activation and production of pro-inflammatory cytokines by TLR4 stimulation, compared to p62 +/+ MEF cells. Additionally, the TLR4-induced activation of signal transduction was significantly augmented in p62 -/- MEF cells, indicating that p62 was negatively implicated in TLR4-mediated signaling. Biochemical studies revealed that p62 interacted with the internal domain of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which is critical for associating with the TNF receptor associated factor 6 (TRAF6)-ECSIT complex to activate NF-κB in TLR4 signaling. Interestingly, p62-ECSIT interaction inhibited the interaction between TRAF6 and ECSIT and attenuated the ubiquitination of ECSIT. Furthermore, upon LPS challenge, the mortality of p62 -/- (p62-knockout) mice was markedly enhanced compared to p62 +/+ (p62 wild-type) mice. Taken together, our data demonstrate that p62 negatively regulated TLR4 signaling via functional regulation of the TRAF6-ECSIT complex.

Endotoxin tolerance develops in the late phase of sepsis to protect cells from an early hyperinflammatory response. Nonetheless, because it induces an immunosuppressive environment, patients with sepsis in its late phase are affected by secondary infections, particularly bacterial pneumonia. Here, we showed that induction of endoplasmic reticulum (ER) stress leads to activation of glycogen synthase kinase 3β (GSK-3β) and X-box-binding protein 1 (XBP-1) in an inositol-requiring enzyme 1α (IRE1α)-mediated manner, which in turn restores the inflammatory response in endotoxin-tolerant macrophages. Animal and in vitro models of endotoxin tolerance were studied along with a model of LPS-induced endotoxin tolerance and a model of cecal ligation and puncture (CLP)-induced endotoxin tolerance. To detect the suppressed inflammatory response during endotoxin tolerance, inflammatory-cytokine expression levels were measured by quantitative real-time PCR and an ELISA. Our research revealed that induction of ER stress alleviated lung injury in a septic host infected with Pseudomonas aeruginosa via the activation of GSK-3β and XBP-1 in an IRE1α-mediated manner. Consequently, in the lungs of the septic host infected with P. aeruginosa, symptoms of pneumonia improved and the infecting bacteria were cleared. Thus, for septic patients, determination of immune status may guide the selection of appropriate immunomodulation, and ER stress can be a novel therapeutic strategy restoring the immune response in patients with endotoxin tolerance.

Huntington's disease (HD) is a devastating neurodegenerative disorder, which is caused by the expression and aggregation of polyQ-expanded mutant huntingtin protein (mtHTT). While toxic mtHTT aggregates are primarily eliminated through autophagy, autophagy dysfunction is often observed in HD pathogenesis. Here, we show that ectodermal-neural cortex 1 (ENC1), a novel binding partner of sequestosome 1 (p62), negatively regulates autophagy under endoplasmic reticulum (ER) stress. We found that ER stress significantly increases the expression of ENC1 via inositol-requiring enzyme 1 (IRE1)-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) pathway. Ectopic expression of ENC1 alone induces the accumulation of detergent-resistant mtHTT aggregates and downregulation of ENC1 alleviates ER stress-induced mtHTT aggregation. Simultaneously, ER stress-induced impairment of autophagy flux is ameliorated by downregulation of ENC1. From immunoprecipitation and immunocytochemical assays, we found that ENC1 binds to p62 through its BTB and C-terminal Kelch (BACK) domain and this interaction is enhanced under ER stress. In particular, ENC1 preferentially interacts with the phosphorylated p62 at Ser403 during ER stress. Interestingly, ENC1 colocalizes with mtHTT aggregates and its C-terminal Kelch domain is required for interfering with the access of p62 to ubiquitinated mtHTT aggregates, thus inhibiting cargo recognition of p62. Accordingly, knockdown of ENC1 expression enhances colocalization of p62 with mtHTT aggregates. Consequently, ENC1 knockdown relieves death of neuronal cells expressing mtHTT under ER stress. These results suggest that ENC1 interacts with the phosphorylated p62 to impair autophagic degradation of mtHTT aggregates and affects cargo recognition failure under ER stress, leading to the accumulation and neurotoxicity of mtHTT aggregates.

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory responses remain largely unknown. The NLRP3 (NLR family, pyrin domain containing 3) inflammasome serves as a platform that triggers the activation of CASP1 (caspase 1) and secretion of proinflammatory cytokines. Here, we demonstrate that SESN2 (sestrin 2), known as stress-inducible protein, suppresses prolonged NLRP3 inflammasome activation by clearance of damaged mitochondria through inducing mitophagy in macrophages. SESN2 plays a dual role in inducing mitophagy in response to inflammasome activation. First, SESN2 induces “mitochondrial priming” by marking mitochondria for recognition by the autophagic machinery. For mitochondrial preparing, SESN2 facilitates the perinuclear-clustering of mitochondria by mediating aggregation of SQSTM1 (sequestosome 1) and its binding to lysine 63 (Lys63)-linked ubiquitins on the mitochondrial surface. Second, SESN2 activates the specific autophagic machinery for degradation of primed mitochondria via an increase of ULK1 (unc-51 like kinase 1) protein levels. Moreover, increased SESN2 expression by extended LPS (lipopolysaccharide) stimulation is mediated by NOS2 (nitric oxide synthase 2, inducible)-mediated NO (nitric oxide) in macrophages. Thus, Sesn2-deficient mice displayed defective mitophagy, which resulted in hyperactivation of inflammasomes and increased mortality in 2 different sepsis models. Our findings define a unique regulatory mechanism of mitophagy activation for immunological homeostasis that protects the host from sepsis.

Event Abstract Back to Event ER Stress Sensitizes TLR Signaling and Desensitizes LPS Tolerance through IRE1a-mediated Activation of GSK-3b Sena Kim1, Yeonsoo Joe1, Jaekyoon Shin2, Stefan W. Ryter3 and Hun Taeg Chung1* 1 University of Ulsan, Republic of Korea 2 School of Medicine and Samsung Biomedical Research Institute, Sungkyunkwan University, Republic of Korea 3 Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, United States Tolerance to endotoxin that is triggered by prior exposure to toll-like receptor (TLR) lignads, provides a mechanism with which to dampen inflammatory cytokines. The ER stress activates GSK-3b to regulate the expression of inflammatory cytokines. Here we found that ER stress induces stimulation of IRE1a-mediated tyrosine phosphorylation and serine dephosphorylation of GSK-3b, which synergistically activates TLR signaling for cytokine production and obliterates endotoxin tolerance. Furthermore, activated GSK-3b increases the abundance of nuclear coactivator, RIP140 which enhances proinflammatory cytokine production and decreases anti-inflammatory cytokine production. Our results identify an unsuspected critical role for IRE1a-mediated GSK-3b activation in inflammation and endotoxin tolerance. Acknowledgements This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2012M3A9C3048687). Keywords: Toll-Like Receptor 4, Endotoxin tolerance, GSK-3, er stress, Inflammation Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Kim S, Joe Y, Shin J, Ryter SW and Chung H (2013). ER Stress Sensitizes TLR Signaling and Desensitizes LPS Tolerance through IRE1a-mediated Activation of GSK-3b. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01143 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Hun Taeg Chung, University of Ulsan, Ulsan, Republic of Korea, chung@ulsan.ac.kr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sena Kim Yeonsoo Joe Jaekyoon Shin Stefan W Ryter Hun Taeg Chung Google Sena Kim Yeonsoo Joe Jaekyoon Shin Stefan W Ryter Hun Taeg Chung Google Scholar Sena Kim Yeonsoo Joe Jaekyoon Shin Stefan W Ryter Hun Taeg Chung PubMed Sena Kim Yeonsoo Joe Jaekyoon Shin Stefan W Ryter Hun Taeg Chung Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.

Berberine exerts an anti-adipogenic activity that is associated with the down-regulation of C/EBPα and PPARγ. Stimulation of AMP-activated kinase (AMPK) caused by inhibition of mitochondrial respiration has been suggested to underlie such molecular regulation. In the present study, we show that berberine up-regulated the expression of two different sets of C/EBP inhibitors, CHOP and DEC2, while down-modulating C/EBPα, PPARγ and other adipogenic markers and effectors in differentiating 3T3-L1 preadipocytes and mature adipocytes. Data also suggested that the berberine-induced up-regulation of CHOP and DEC2 was attributable to selective activation of an unfolded protein response (UPR) and modified extracellular environment, respectively. As a result, the anti-adipogenic activity of berberine was diminished remarkably by adjusting the differentiation culture media and limitedly but consistently by knockdown of CHOP expression. Together, up-regulation of C/EBP inhibitors appears to underlie the berberine-induced repression of C/EBPα and PPARγ and, so, the inhibition of adipogenesis.

Prolonged mitosis due to aberrant chromosome segregation permits cells to enter the G1 phase without cytokinesis and subsequently triggers the p53-dependent cell death program, known as mitotic catastrophe. Cells which fail to go through mitotic catastrophe create aneuploidy, posing a risk of oncogenesis. In the present report, we show that p62-mediated non-canonical activation of Nrf2 leads to the persistent expression of Nqo1, which plays a critical role for p53 stabilization during mitotic catastrophe. With prolonged exposure to nocodazole, a microtubule-depolymerizing agent, p62-deficient HCT116 cells exhibited an accumulation of a polyploid population with a limited appearance of apoptotic cells, which was attributable to the attenuated stabilization of p53. Combinatorial gene manipulation analysis verified that the regulatory cascade with a hierarchy of p62–Keap1–Nrf2–Nqo1 is required for p53 stabilization for mitotic catastrophe. This is consistent with the role of Nqo1 as a gatekeeper for proteasomal degradation of p53. Thus, we demonstrate for the first time the functional connection between the non-canonical Nrf2 pathway and p53-dependent cell death program upon prolonged mitosis.

As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.

The T cell-specific tyrosine kinase, p56(lck), plays crucial roles in T cell receptor (TCR)-mediated T cell activation. Here, we report that SOCS-6 (suppressor of cytokine signaling-6) is a negative regulator of p56(lck). SOCS-6 was identified as a protein binding to the kinase domain of p56(lck) through yeast two-hybrid screening. SOCS-6 bound specifically to p56(lck) (F505), which mimics the active form of p56(lck), but not to wild type p56(lck). In Jurkat T cells, SOCS-6 binding to p56(lck) was detected 1-2 h after TCR stimulation. Confocal microscopy showed that upon APC-T cell conjugation, SOCS-6 was recruited to the immunological synapse and colocalized with the active form of p56(lck). SOCS-6 promoted p56(lck) ubiquitination and its subsequent targeting to the proteasome. Moreover, SOCS-6 overexpression led to repression of TCR-dependent interleukin-2 promoter activity. These results establish that SOCS-6 acts as a negative regulator of T cell activation by promoting ubiquitin-dependent proteolysis.

2 Laboratory of Molecular Cell Biology, Sungkyunkwan University School of Medicine and Samsung Biomedical Research Institute, Suwon, Kyonggi-Do 440-746, Republic of Korea Running head: SOCS-6 as a negative regulator of T cell activation Address correspondence to Dr. Yungdae Yun, Department of Life Science, Ewha Woman’s University, 111 Daehyundong, Seodaemungu, 120-750, Seoul, Republic of Korea. Tel) 822-3277-3248, Fax) 822-32773760, E-mail) YUNYUNG@ewha.ac.kr

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia. because of abnormal turnover and function of cellular organelles.

The capsid protein of the West Nile virus (WNV) functions as an apoptotic agonist via the induction of mitochondrial dysfunction and the activation of caspases-9 and -3. Here, we have determined that the WNV capsid (WNVCp) is capable of binding to and sequestering HDM2 into the nucleolus. WNVCp was shown to interfere with the formation of the HDM2 and p53 complex, thereby causing the stabilization of p53 and the subsequent induction of its target apoptotic protein, Bax. Whereas WNVCp was capable of inducing the p53-dependent apoptotic process in wild-type mouse embryonic fibroblasts (MEF) or SH-SY5Y cells, it exerted no significant effects on p53-null MEF or on p53-knockdown SH-SY5Y cells. This suggests that WNVCp-mediated apoptosis requires p53. Furthermore, when WNV was transfected into cells, endogenous Hdm2 and WNVCp were able to interact physically. WNVCp expressed in wild-type MEF proved able to induce the translocation of the endogenous Hdm2 into the nucleolus. Consistently, WNV was highly pathogenic in the presence of p53, and was less so in the absence of p53. The results of these studies suggest that the apoptotic mechanism mediated by WNV might occur in accordance in a fashion similar to that of the tumour-suppressing mechanism mediated by ARF.

We have developed fluorescence polarization (FP) assays of human melanocortin 4 receptor (MC4R) in 384-well microtiter plates using TAMRA-NDP-MSH as a tracer. The rank order of potency of agonists and antagonists agrees well relative to the published assays: SHU9119>MTII>NDP alphaMSH>alphaMSH. We have screened libraries of Korean plant extracts and frog peptide analogues in search of MC4R ligands using FP assays and cell-based CRE luciferase reporter assays. We report that FLGFLFKVASK, FLGWLFKVASK, FLGALFKWASK, and FLGWLFKWASK are the peptide analogues, which bind to human MC4R receptor with good affinity in vitro. FLGWLFKVASK and FLGWLFKWASK stimulated CRE-driven reporter gene via MC4R. In luciferase reporter assays, they possess the pharmacological and functional profiles of full agonists. We demonstrate the interaction of MC4R with 11-residue antimicrobial peptides derived from the Korean frog, Rana rugosa. The results suggest that MC4R interacts promiscuously with bioactive analogues of antimicrobial peptide, gaegurin-5.

During T-cell activation, Ser59 in the unique N-terminal region of p56"k is phosphorylated. Mutation of Ser59 to Glu59 mimics Ser59 phosphorylation, and upon CD4 crosslinking, this mutant p561k induces tyrosine phosphorylation of intracellular proteins distinct from those induced by wild-type pS6kk. Mutant and wild-type pS6lk have similar affinities for CD4 and similar kinase activities. In glutathione S-transferase fusion proteins, the p56kk Src homology 2 (SH2) domain with the SH3 domain and the unique N-terminal region (including Ser-59) has a different binding specificity for phosphotyrosyl proteins than the SH2 domain alone. Either deletion of the unique N-terminal region or mutation of Ser59 to Glu59 in the fusion protein reverts the phosphotyrosyl protein binding specificity back to that of the SH2 domain alone. These results suggest that phosphorylation of Ser59 regulates the function of p56kk by controlling binding specificity of its SH2 domain. The association of p56lck, a lymphoid cell-specific Src-family protein-tyrosine kinase, with the intracellular domains of the CD4 and CD8 molecules (1) and the direct involvement of p56lck in T-cell activation (2), cytolytic activity of cytotoxic T lymphocytes (3), and thymic differentiation (4) have all been described (reviewed in ref. 5). Members of the Src family of cytoplasmic tyrosine kinases, including p561ck, have an Nterminal myristoylation site, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain, and a C-terminal catalytic domain with conserved regulatory tyrosine phosphorylation sites (6). SH2 and SH3 domains mediate protein-protein interactions by binding to proteins containing phosphotyrosine and a proline-rich region, respectively (7). The first 66 aa of p56Ick are unrelated in amino acid sequence to similarly placed unique regions in other members of the Src family (6). Tyrosine kinase activity of p561ck is suppressed by phosphorylation of the C-terminal inhibitory tyrosine residue (Tyr505) that is catalyzed by another tyrosine kinase, Csk (8). The catalytic activity of p56kck is upregulated by a CD45-mediated dephosphorylation of Tyr505 (9) and an increase in the autophosphorylation of Tyr394 (10). It has been proposed that phosphorylated C-terminal Tyr505 binds intramolecularly to the SH2 domain, blocking the kinase domain from its substrate (5, 11). In this model, dephosphorylated Tyr505 is released from the SH2 domain, and the SH2 domain becomes free for binding to other signaling proteins. Although SH2 domains are structurally homologous, those from different proteins have distinct binding specificities for particular phosphotyrosyl proteins, determined by the amino acid sequence surrounding the phosphotyrosine residue (12). Many of the complexes formed by binding of SH2 domains to phosphotyrosine-containing proteins have been described as The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. having critical roles in signal transduction (7), including the Ras signal pathway (13). As the N and C termini of the SH2 domain are in close proximity on opposite sides of the phosphotyrosine-binding region in the three-dimensional structure, it has been suggested that other regions of these proteins may not significantly affect function and folding of the SH2 domain (14,15). However, some binding studies suggest that SH2 domains can be intramolecularly modified by other regions of these SH2 domain-containing proteins (16, 17). Regulatory events for the p56kk SH2 domain could also take place in the context of the full-length protein. Transient phosphorylation of Ser59 in the unique N-terminal region of p56kck by a proline-directed kinase [most likely by mitogen-activated protein kinase (MAP kinase)] during T-cell activation has been observed (refs. 18 and 19; unpublished work). Phosphorylation at this site induces a shift in apparent molecular mass of p561ck from 56 kDa to 61 kDa in SDS/ polyacrylamide gels. We demonstrate that the Ser59 phosphorylation site regulates p561ck by modifying the binding affinity and specificity of the p561ck SH2 domain for phosphotyrosinecontaining proteins. MATERIALS AND METHODS Site-Directed Mutagenesis, Expression, and Activation of Cells. Site-directed mutagenesis was performed on uracilcontaining phage DNA (20). HeLa and CD4+ HeLa cells (21) and Jurkat T cells were maintained in 10% fetal bovine serum-supplemented Dulbecco's modified Eagle's medium and RPMI 1640, respectively. HeLa cells were transiently transfected with 20 ,ug of cDNA in the pCDNA-1 expression vector (Invitrogen) per 10-cm plate by the calcium phosphate precipitation method (22) and used for assays 48-72 hr later. v-src transformation was induced by transient expression of v-src. For antibody crosslinking, OKT4 and T3b monoclonal antibodies were used to activate cells through CD4 and CD3, respectively. Cells were incubated at 4°C for 30 min with primary antibody and washed twice at 4°C. Crosslinking was initiated by the addition of goat anti-mouse IgG antibody at 37°C and terminated by washing with ice-cold phosphatebuffered saline or by boiling in SDS/PAGE loading buffer. Production of Glutathione S-Transferase (GST) Fusion Proteins. GST fusion proteins were produced as described (23). Portions of the lck gene were isolated by PCR and were subcloned into the Nde I and EcoRI sites ofpGEX-3Xb vector. When the bacteria bearing the vector reached an OD555 of 0.6, protein expression was induced by adding 1 mM isopropyl 13-D-thiogalactopyranoside for 3 hr. Bacterial pellets were lysed by sonication in 10 mM Hepes, pH 7.5/150 mM NaCl/0.5% (vol/vol) Triton X-100/10 mM dithiothreitol. The lysate was cleared by centrifugation at 15,000 rpm in a Sorvall SS34 rotor for 30 min. GST fusion proteins were purified on glutathioneAbbreviations: SH, Src homology; GST, glutathione S-transferase; MAP kinase, mitogen-activated protein kinase.

Schisandra chinensis Baill. (Shisandraceae) is used commonly in traditional Korean herbal medicine. One new dibenzocyclooctene lignan compound, benzoylgomisin Q, was found, for the first time, in the fruits of Schisandra chinensis Baill., together with five known lignan compounds; schisandrin, gomisin A, gomisin G, deoxyschisandrin, and gomisin N, using an ESI-positive MS-preparative HPLC auto-purification system. The structures of these compounds were determined via UV, MS, and NMR spectral studies.