Subcellular Golgi localization of stathmin family proteins is promoted by a specific set of DHHC palmitoyl transferases.
ABSTRACT: Protein palmitoylation is a reversible lipid modification that plays critical roles in protein sorting and targeting to specific cellular compartments. The neuronal microtubule-regulatory phosphoproteins of the stathmin family (SCG10/stathmin 2, SCLIP/stathmin 3, and RB3/stathmin 4) are peripheral proteins that fulfill specific and complementary roles in the formation and maturation of the nervous system. All neuronal stathmins are localized at the Golgi complex and at vesicles along axons and dendrites. Their membrane anchoring results from palmitoylation of two close cysteine residues present within their homologous N-terminal targeting domains. By preventing palmitoylation with 2-bromopalmitate or disrupting the integrity of the Golgi with brefeldin A, we were able to show that palmitoylation of stathmins 2 and 3 likely occurs at the Golgi and is crucial for their specific subcellular localization and trafficking. In addition, this membrane binding is promoted by a specific set of palmitoyl transferases that localize with stathmins 2 and 3 at the Golgi, directly interact with them, and enhance their membrane association. The subcellular membrane-associated microtubule-regulatory activity of stathmins might then be fine-tuned by extracellular stimuli controlling their reversible palmitoylation, which can be viewed as a crucial regulatory process for specific and local functions of stathmins in neurons.
Project description:In vertebrates, stathmins form a family of proteins possessing two tubulin binding repeats (TBRs), which each binds one soluble tubulin heterodimer. The stathmins thus sequester two tubulins in a phosphorylation-dependent manner, providing a link between signal transduction and microtubule dynamics. In Drosophila, we show here that a single stathmin gene (stai) encodes a family of D-stathmin proteins. Two of the D-stathmins are maternally deposited and then restricted to germ cells, and the other two are detected in the nervous system during embryo development. Like in vertebrates, the nervous system-enriched stathmins contain an N-terminal domain involved in subcellular targeting. All the D-stathmins possess a domain containing three or four predicted TBRs, and we demonstrate here, using complementary biochemical and biophysical methods, that all four predicted TBR domains actually bind tubulin. D-stathmins can indeed bind up to four tubulins, the resulting complex being directly visualized by electron microscopy. Phylogenetic analysis shows that the presence of regulated multiple tubulin sites is a conserved characteristic of stathmins in invertebrates and allows us to predict key residues in stathmin for the binding of tubulin. Altogether, our results reveal that the single Drosophila stathmin gene codes for a stathmin family similar to the multigene vertebrate one, but with particular tubulin binding properties.
Project description:Pathological Golgi fragmentation represents a constant pre-clinical feature of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) but its molecular mechanisms remain hitherto unclear.Here, we show that the severe Golgi fragmentation in transgenic mutant SOD1(G85R) and SOD1(G93A) mouse motor neurons is associated with defective polymerization of Golgi-derived microtubules, loss of the COPI coat subunit ?-COP, cytoplasmic dispersion of the Golgi tether GM130, strong accumulation of the ER-Golgi v-SNAREs GS15 and GS28 as well as tubular/vesicular Golgi fragmentation. Data mining, transcriptomic and protein analyses demonstrate that both SOD1 mutants cause early presymptomatic and rapidly progressive up-regulation of the microtubule-destabilizing proteins Stathmins 1 and 2. Remarkably, mutant SOD1-triggered Golgi fragmentation and Golgi SNARE accumulation are recapitulated by Stathmin 1/2 overexpression but completely rescued by Stathmin 1/2 knockdown or the microtubule-stabilizing drug Taxol.We conclude that Stathmin-triggered microtubule destabilization mediates Golgi fragmentation in mutant SOD1-linked ALS and potentially also in related motor neuron diseases.
Project description:Secretion of proteins and peptides from eukaryotic cells takes place by both constitutive and regulated pathways. Regulated secretion may involve interplay of proteins that are currently unknown. Recent studies suggest an important role of chromogranin A (CHGA) in the regulated secretory pathway in neuroendocrine cells, but the mechanism by which CHGA enters the regulated pathway, or even triggers the formation of the pathway, remains unclear. In this study, we used a transcriptome/proteome-wide approach, to discover binding partners for CHGA, by employing a phage display cDNA library method. Several proteins within or adjacent to the secretory pathway were initially detected as binding partners of recombinant human CHGA. We then focused on the trans-Golgi protein SCLIP (STMN3) and its stathmin paralog SCG10 (STMN2) for functional study. Co-immunoprecipitation experiments confirmed the interaction of each of these two proteins with CHGA in vitro. SCLIP and SCG10 were colocalized to the Golgi apparatus of chromaffin cells in vivo and shared localization with CHGA as it transited the Golgi. Downregulation of either SCLIP or SCG10 by synthetic siRNAs virtually abolished chromaffin cell secretion of a transfected CHGA-EAP chimera (expressing CHGA fused to an enzymatic reporter, and trafficked to the regulated pathway). SCLIP siRNA also decreased the level of secretion of endogenous CHGA and SCG2, as well as transfected human growth hormone, while SCG10 siRNA decreased the level of regulated secretion of endogenous CHGB. Moreover, a dominant negative mutant of SCG10 (Cys 22,Cys 24-->Ala 22,Ala 24) significantly blocked secretion of the transfected CHGA-EAP chimera. A decrease in the buoyant density of chromaffin granules was observed after downregulation of SCG10 by siRNA, suggesting participation of these stathmins in granule formation or maturation. We conclude that SCLIP and SCG10 interact with CHGA, share partial colocalization in the Golgi apparatus, and may be necessary for typical transmitter storage and release from chromaffin cells.
Project description:Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.
Project description:MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.
Project description:In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubule-regulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.
Project description:SCLIP, a microtubule-destabilizing phosphoprotein, is known to be involved in the development of the central nervous system (CNS). It has been well established that there are notable parallels between normal development and tumorigenesis, especially in glioma. However, no studies have examined the significance of SCLIP in gliomagenesis. To address this, we investigated the expression of SCLIP and its roles in the development of gliomas. Notably, we found that SCLIP was highly expressed in various grades of glioma samples, as compared with normal brain tissues. Overexpression of SCLIP dramatically stimulated tumor cell migration and invasion as well as proliferation and downregulation of SCLIP showed opposite effects, establishing an important oncogenic role for this gene. Furthermore, we revealed that STAT3 was required to maintain SCLIP stability, suggesting that overexpression of STAT3 may be a critical step to facilitate microtubule dynamics and subsequently promotes migration and invasion of glioma cells. Taken together, our findings demonstrate that SCLIP plays an important role in glioma pathology, and may represent a novel therapeutic strategy against human glioma.
Project description:BACKGROUND: Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells. RESULTS: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism. CONCLUSION: In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.
Project description:c-Jun NH(2)-terminal kinases (JNKs) are essential during brain development, when they regulate morphogenic changes involving cell movement and migration. In the adult, JNK determines neuronal cytoarchitecture. To help uncover the molecular effectors for JNKs in these events, we affinity purified JNK-interacting proteins from brain. This revealed that the stathmin family microtubule-destabilizing proteins SCG10, SCLIP, RB3, and RB3' interact tightly with JNK. Furthermore, SCG10 is also phosphorylated by JNK in vivo on sites that regulate its microtubule depolymerizing activity, serines 62 and 73. SCG10-S73 phosphorylation is significantly decreased in JNK1-/- cortex, indicating that JNK1 phosphorylates SCG10 in developing forebrain. JNK phosphorylation of SCG10 determines axodendritic length in cerebrocortical cultures, and JNK site-phosphorylated SCG10 colocalizes with active JNK in embryonic brain regions undergoing neurite elongation and migration. We demonstrate that inhibition of cytoplasmic JNK and expression of SCG10-62A/73A both inhibited fluorescent tubulin recovery after photobleaching. These data suggest that JNK1 is responsible for regulation of SCG10 depolymerizing activity and neurite elongation during brain development.
Project description:Palmitoylation is a posttranslational modification that regulates protein trafficking and stability. In this study we investigated whether the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins syntaxin 7 and syntaxin 8 are modified with palmitate. Using metabolic labeling and site-directed mutagenesis, we show that human syntaxins 7 and 8 are modified with palmitate through a thioester linkage. Palmitoylation is dependent upon cysteine 239 of human syntaxin 7 and cysteine 214 of syntaxin 8, residues that are located on the cytoplasmic face of the transmembrane domain (TMD). Palmitoylation of syntaxin 8 is minimally affected by the Golgi-disturbing agent brefeldin A (BFA), whereas BFA dramatically inhibits palmitoylation of syntaxin7. The differential effect of BFA suggests that palmitoylation of syntaxins 7 and 8 occurs in distinct subcellular compartments. Palmitoylation does not affect the rate of protein turnover of syntaxins 7 and 8 nor does it influence the steady-state localization of syntaxin 8 in late endosomes. Syntaxin 7 actively cycles between endosomes and the plasma membrane. Palmitoylation-defective syntaxin 7 is selectively retained on the plasma membrane, suggesting that palmitoylation is important for intercompartmental transport of syntaxin 7.