Linyi Chen

The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase

Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.

Heterotypic Gap Junctions between Two Neurons in the Drosophila Brain Are Critical for Memory

Gap junctions play an important role in the regulation of neuronal metabolism and homeostasis by serving as connections that enable small molecules to pass between cells and synchronize activity between cells. Although recent studies have linked gap junctions to memory formation, it remains unclear how they contribute to this process. Gap junctions are hexameric hemichannels formed from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates. Here we show that two modulatory neurons, the anterior paired lateral (APL) neuron and the dorsal paired medial (DPM) neuron, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNA interference-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons, respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM.

Ubiquitin-independent entry into the yeast recycling pathway

The yeast a‐factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand‐independent pathway links to vacuolar degradation of the receptor, while a ligand‐dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand‐dependent endocytosis mechanism. The doa4Δ mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand‐dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand‐dependent uptake was further indicated by analysis of receptor mutants having Lys‐to‐Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand‐induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4‐dependent turnover of the receptor (for receptor internalized via the ligand‐dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys‐to‐Arg mutations was noted. Following a prolonged exposure of the cells to the a‐factor ligand, rather than being delivered to the vacuolar lumen, the Lys‐to‐Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a‐factor‐dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.

A network map of FGF-1/FGFR signaling system

Fibroblast growth factor-1 (FGF-1) is a well characterized growth factor among the 22 members of the FGF superfamily in humans. It binds to all the four known FGF receptors and regulates a plethora of functions including cell growth, proliferation, migration, differentiation, and survival in different cell types. FGF-1 is involved in the regulation of diverse physiological processes such as development, angiogenesis, wound healing, adipogenesis, and neurogenesis. Deregulation of FGF-1 signaling is not only implicated in tumorigenesis but also is associated with tumor invasion and metastasis. Given the biomedical significance of FGFs and the fact that individual FGFs have different roles in diverse physiological processes, the analysis of signaling pathways induced by the binding of specific FGFs to their cognate receptors demands more focused efforts. Currently, there are no resources in the public domain that facilitate the analysis of signaling pathways induced by individual FGFs in the FGF/FGFR signaling system. Towards this, we have developed a resource of signaling reactions triggered by FGF-1/FGFR system in various cell types/tissues. The pathway data and the reaction map are made available for download in different community standard data exchange formats through NetPath and NetSlim signaling pathway resources.

Electrical stimulation promotes nerve growth factor-induced neurite outgrowth and signaling

BACKGROUND Neurotrophins are important regulators for neural development and regeneration. Nerve growth factor (NGF) therapy has been tested in various models of neural injury and degeneration. However, whether NGF can reach target tissues and maintain effective concentration for a certain period of time remains uncertain. To facilitate neural regeneration, we investigate the possibility of combining NGF and electrical stimulation (ES) in promoting neurite outgrowth, an essential process during neural regeneration. METHODS PC12 cells were seeded on collagen and indium tin oxide (ITO)-coated area on the transparent conductive devices. Cells were then subjected to the combination of ES and NGF treatment. Neurite outgrowth was compared. RESULTS Our findings suggest that ES of 100mV/mm together with NGF provides optimal effect on neurite outgrowth of PC12 cells. ES increases NGF-induced neurite length but reduces neurite branching, indicative of its primary effect on neurite elongation instead of initiation. One mechanism that ES enhances neurite outgrowth is through increasing NGF-induced phosphorylation of ERK1/2 (pERK1/2) and expression of Egr1 gene. ES has previously been demonstrated to increase the activity of protein kinase C (PKC). Our result indicates that activating PKC further increases NGF-induced pERK1/2 and thus neurite outgrowth. CONCLUSION It is likely that ES promotes NGF-induced neurite outgrowth through modulating the activity of ERK1/2. GENERAL SIGNIFICANCE Findings from this study suggest that combining ES and NGF provides a promising strategy for promoting neurite outgrowth.

SH2B1β enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway

Genetic studies have established the crucial roles of FGF signaling, FGF-induced gene expression and morphogenesis during embryogenesis. In this study, we showed that overexpressing a signaling adaptor protein, SH2B1beta, enhanced FGF1-induced neurite outgrowth in PC12 cells. SH2B1beta has previously been shown to promote nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF)-induced neurite outgrowth, in part, through prolonging NGF and GDNF-induced signaling. To delineate how SH2B1beta promotes FGF1-induced neurite outgrowth, we examined its role in FGF1-dependent signaling. Our data suggest that SH2B1beta enhances and prolongs FGF1-induced MEK-ERK1/2 and PI3K-AKT pathways. We also provided the first evidence that FGF1 induces the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at serine 727 [pSTAT3(S727)] in PC12 cells. SH2B1beta enhances this phosphorylation and the expression of the immediate early gene, Egr1. Through inhibitor assays, we have further shown that MEK-ERK1/2 is required for FGF1-induced neurite outgrowth, pSTAT3(S727) and Egr1 expression. Moreover, inhibiting Rho kinase, ROCK, enhances FGF1-induced neurite outgrowth through pSTAT3(S727)-independent manner. Taken together, our results demonstrate, for the first time, that SH2B1beta enhances FGF1-induced neurite outgrowth in PC12 cells mainly through MEK-ERK1/2-STAT3-Egr1 pathway.

Adapter Protein SH2-Bβ Undergoes Nucleocytoplasmic Shuttling: Implications for Nerve Growth Factor Induction of Neuronal Differentiation

ABSTRACT The adapter protein SH2-B has been shown to bind to activated nerve growth factor (NGF) receptor TrkA and has been implicated in NGF-induced neuronal differentiation and the survival of sympathetic neurons. However, the mechanism by which SH2-B enhances and maintains neurite outgrowth is unclear. We examined the ability of truncation mutants to regulate neuronal differentiation and observed that certain truncation mutants localized in the nucleus rather than in the cytoplasm or at the plasma membrane as reported for wild-type SH2-Bβ. Addition of the nuclear export inhibitor leptomycin B caused both overexpressed wild-type and endogenous SH2-Bβ to accumulate in the nucleus of both PC12 cells and COS-7 cells as did deletion of a putative nuclear export sequence (amino acids 224 to 233) or mutation of two critical lysines in that sequence. Deleting or mutating the nuclear export signal caused SH2-Bβ to lose its ability to enhance NGF-induced differentiation of PC12 cells. Neither the NGF-induced phosphorylation of ERKs 1 and 2 nor their subcellular distribution was altered in PC12 cells stably expressing the nuclear export-defective SH2-Bβ(L231A, L233A). These data provide strong evidence that SH2-Bβ shuttles constitutively between the nucleus and cytoplasm. However, SH2-Bβ needs continuous access to the cytoplasm and/or plasma membrane to participate in NGF-induced neurite outgrowth. These data also suggest that the stimulatory effect of SH2-Bβ on NGF-induced neurite outgrowth of PC12 cells is either downstream of ERKs or via some other pathway yet to be identified.

SH2-B Is a Positive Regulator of Nerve Growth Factor-mediated Activation of the Akt/Forkhead Pathway in PC12 Cells

To gain insight into the mechanism by which the adapter protein SH2-B promotes nerve growth factor (NGF)-mediated neuronal differentiation and survival, the effect of SH2-B on the serine/threonine kinase Akt/protein kinase B and downstream effector proteins was examined. PC12 cells stably overexpressing SH2-Bβ, which exhibit enhanced NGF-induced neuronal differentiation compared with control cells, showed enhanced and prolonged NGF-induced phosphorylation of Akt on Ser473 and Akt enzymatic activity. Surprisingly, NGF-induced phosphorylation of Akt on Ser473 and Akt activity were not altered in cells overexpressing SH2-Bβ(R555E) with a defective SH2 domain, despite the ability of the overexpressed SH2-Bβ(R555E) to block NGF-induced differentiation. Consistent with SH2-Bβ enhancing the activity of Akt, cells overexpressing SH2-Bβ but not SH2-Bβ(R555E) exhibited increased and/or prolonged phosphorylation of the pro-apoptotic Akt effector proteins, glycogen synthase kinase-3, and forkhead transcription factors, FKHRL1/FOXO3 and FKHR/FOXO1. Immunolocalization studies indicated that, although ectopically expressed FKHR was primarily concentrated in the cytoplasm of control cells and cells transiently overexpressing SH2-Bβ, it was concentrated in the nucleus of cells transiently overexpressing SH2-Bβ(R555E). Similarly, SH2-Bβ stimulated the accumulation of FKHR in the cytoplasm of 293T and COS-7 cells, whereas SH2-Bβ(R555E) enhanced its accumulation in the nucleus. In PC12 cells stably expressing forms of SH2-Bβ, SH2-Bβ mimicked the ability of NGF to promote redistribution of FKHR to the cytoplasm whereas SH2-Bβ(R555E) blocked this effect of NGF. Taken together, these data indicate that SH2-B is a positive regulator of NGF-mediated activation of the Akt/Forkhead pathway.

Cholestane-3β, 5α, 6β-triol Suppresses Proliferation, Migration, and Invasion of Human Prostate Cancer Cells

Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.

Nucleocytoplasmic Shuttling of the Adapter Protein SH2B1β (SH2-Bβ) Is Required for Nerve Growth Factor (NGF)-Dependent Neurite Outgrowth and Enhancement of Expression of a Subset of NGF-Responsive Genes

The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1s function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1beta. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1beta from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1beta nuclear import also prevents SH2B1beta from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1beta nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1beta enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1beta must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.