Role of a tyrosine phosphorylation of SMG-9 in binding of SMG-9 to IQGAP and the NMD complex.

SMG-9 is a component of the NMD complex, a heterotetramer that also includes SMG-1 and SMG-8 in the complex. SMG-9 was also originally identified as a tyrosine-phosphorylated protein but the role of the phosphorylation is not yet known. In this study, we determined that IQGAP protein, an actin cytoskeleton modifier acts as a binding partner with SMG-9 and this binding is regulated by phosphorylation of SMG-9 at Tyr-41. SMG-9 is co-localized with IQGAP1 as a part of the process of actin enrichment in non-stimulated cells, but not in the EGF-stimulated cells. Furthermore, an increase in the ability of SMG-9 to bind to SMG-8 occurs in response to EGF stimulation. These results suggest that tyrosine phosphorylation of SMG-9 may play a role in the formation of the NMD complex in the cells stimulated by the growth factor.

Proteomic analysis for neuronal vacuolation induced by MK-801 in rat retrosplenial cortex.

Proteomic analysis was carried out for neuronal vacuolation in rat retrosplenial cortex (RSC) induced by MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist. Female rats were given a single subcutaneous (sc) injection of either MK-801 (9 mg/kg in saline) or saline. Comparison of changes in proteins in the RSC region between MK-801- and saline-treated groups revealed that MK-801 induced changes in six proteins involved in vesicular transport (vesicle-fusing ATPase) and glycolysis (fructose-bisphosphate aldolase C, triosephosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase).

Induction of endosomal/lysosomal pathways in differentiating osteoblasts as revealed by combined proteomic and transcriptomic analyses.

We have analyzed proteome changes associated with bone-forming osteoblast differentiation by quantitative differential proteomic and transcriptomic analyses using in vitro differentiation model. Sixty nine proteins were found up-regulated (>2-fold) and 18 were down-regulated (<0.5-fold) at protein level. The mRNA levels of these proteins were then analyzed by quantitative real-time PCR combined with clustering analysis. The most prominent cluster with increased protein and mRNA levels contains endosomal and lysosomal proteins, demonstrating the drastic induction of degradative endosomal/lysosomal pathways in osteoblasts. Osteoblasts, therefore, are involved not only in the synthesis but also in the turnover of the extracellular matrix proteins such as collagens.

Distinct functions of human MVB12A and MVB12B in the ESCRT-I dependent on their posttranslational modifications.

ESCRT-I, which mediates the sorting of ubiquitinated cargo protein from the plasma membrane to the endosomal vesicle, comprises a heterotetramer of TSG101 (Vps23), Vps28, Vps37 and MVB12 protein. In humans, the structurally similar subtypes MVB12A and MVB12B are subunits of ESCRT-I. However, no functional description of these proteins has been described. Here we show the differing effects of tyrosine phosphorylation and ubiquitination of both MVB12 proteins on their respective functions. As noted in our previous study, Tyr204 phosphorylation of MVB12A in response to epidermal growth factor (EGF) stimulation affects binding to CD2AP, which regulates the amounts of EGF receptor bound to ESCRT-I. Strikingly, ubiquitination of Lys264 and Lys290 of MVB12B was induced and led to the instability and inclusion of MVB12B in COS-7 cells. These ubiquitinations increased upon EGF stimulation, which was regulated by the phosphorylations of Tyr241 and Tyr243 of MVB12B. Furthermore, MVB12A was also involved in the aggregation-prone proteins of MVB12B. These results suggest that the expression of MVB12B may be normally suppressed through the ubiquitin-proteasome pathway that simultaneously regulates the fate of MVB12A and the functions of ESCRT-I.

[New functional proteins identified by proteomic analysis in the epidermal growth factor receptor-mediated signaling pathway and application for practical use].

To clarify the whole picture of epidermal growth factor (EGF) signaling pathway, we identified proteins from the EGF-stimulated A431 cells by anti-phospho-tyrosine antibody column chromatography. Over 150 proteins were detected including previously unidentified proteins as well as well-studied proteins. Among these proteins, we picked up four proteins that had not been known in EGF signaling pathway and analyzed their functions. We report the functions of these proteins in this article. 1) CFBP interacts with CD2AP family proteins and functions as a key component in downregulation of EGF receptor protein level following EGF stimulation. 2) Ymer is found to be phosphorylated and ubiquitinated upon EGF stimulation, and functions as a regulator for the downregulation and endocytosis of EGF receptor. 3) CLPABP binds to mitochondria-specific phospholipids, cardiolipin, through its PH domain, and its complex includes various proteins related to mRNA metabolism. 4) GAREM is associated with Grb2 and Shp2. Each association affects the ERK activity. Finally, we discuss the possibilities that these proteins can be used as a novel biomarker protein for cancer and other diseases.

GAREM, a novel adaptor protein for growth factor receptor-bound protein 2, contributes to cellular transformation through the activation of extracellular signal-regulated kinase signaling.

Adaptor proteins for the various growth factor receptors play a crucial role in signal transduction through tyrosine phosphorylation. Several candidates for adaptor proteins with potential effects on the epidermal growth factor (EGF) receptor-mediated signaling pathway have been identified by recent phosphoproteomic studies. Here, we focus on a novel protein, GAREM (Grb2-associated and regulator of Erk/MAPK) as a downstream molecule of the EGF receptor. GAREM is phosphorylated at tyrosine 105 and 453 after EGF stimulation. Grb2 was identified as its binding partner, and the proline-rich motifs of GAREM are recognized by the N- and C-terminal SH3 domains of Grb2. In addition, the tyrosine phosphorylations of GAREM are necessary for its binding to Grb2. Because the amino acid sequence surrounding tyrosine 453 is similar to the immunoreceptor tyrosine-based inhibitory motif, Shp2, a positive regulator of Erk, binds to GAREM in this phosphorylation-dependent manner. Consequently, Erk activation in response to EGF stimulation is regulated by the expression of GAREM in COS-7 and HeLa cells, which occurs independent of the presence of other binding proteins, such as Gab1 and SOS, to the activated EGF receptor. Furthermore, the expression of GAREM has an effect on the transformation activity of cultured cells. Together, these findings suggest that GAREM plays a key role in the ligand-mediated signaling pathway of the EGF receptor and the tumorigenesis of cells.

Synaptic activity prompts gamma-secretase-mediated cleavage of EphA4 and dendritic spine formation.

Alzheimer's disease is an age-dependent neurodegenerative disorder that is characterized by a progressive decline in cognitive function. gamma-secretase dysfunction is evident in many cases of early onset familial Alzheimer's disease. However, the mechanism by which gamma-secretase dysfunction results in memory loss and neurodegeneration is not fully understood. Here, we demonstrate that gamma-secretase is localized at synapses and regulates spine formation. We identify EphA4, one of the Ephrin receptor family members, as a substrate of gamma-secretase, and find that EphA4 processing is enhanced by synaptic activity. Moreover, overexpression of EphA4 intracellular domain increases the number of dendritic spines by activating the Rac signaling pathway. These findings reveal a function for EphA4-mediated intracellular signaling in the morphogenesis of dendritic spines and suggest that the processing of EphA4 by gamma-secretase affects the pathogenesis of Alzheimer's disease.

Novel tyrosine phosphorylated and cardiolipin-binding protein CLPABP functions as mitochondrial RNA granule.

We identified a new protein containing the pleckstrin homology (PH) domain through tyrosine phosphoproteomics using epidermal growth factor-stimulated cells. The tandem PH domains of this protein can bind to mitochondria-specific phospholipid, cardiolipin or its dehydro product, phosphatidic acid; therefore, we have designated this protein as cardiolipin and phosphatidic acid-binding protein (CLPABP). In this study, we show that CLPABP is localized on the tubulin network and the mitochondrial surface in the granular form along with other proteins and RNA. The affinity of CLPABP to mitochondria is elevated depending on the extent of tyrosine phosphorylation. The CLPABP complex contains various proteins related to cytoplasmic mRNA metabolism. The unique subcellular localization of CLPABP requires its PH domains and a multifunctional protein, SF2p32, as its binding protein. The CLPABP granule also contains the cytochrome c transcript, which may be mediated by the RNA-binding protein HuR. Immunofluorescence staining reveals that the CLPABP granule is colocalized with cytochrome c and various ribosomal proteins that are present in the CLPABP complex. Therefore, the CLPABP RNA-protein complex may play a role in transporting cytochrome c mRNA and its translated product to the mitochondria.

CFBP is a novel tyrosine-phosphorylated protein that might function as a regulator of CIN85/CD2AP.

To decipher the global network of the epidermal growth factor (EGF) receptor-mediated signaling pathway, a large scale proteomic analysis of tyrosine-phosphorylated proteins was conducted. Here, we focus on characterizing a novel protein, CFBP (CIN85/CD2AP family binding protein), identified in the study. CFBP was found to be phosphorylated at tyrosine 204 upon EGF stimulation, and the CIN85/CD2AP family was identified as a binding partner. A proline-rich motif of CFBP is recognized by one of the three Src-homology 3 domains of CIN85/CD2AP, and the affinity of the interaction is regulated by the tyrosine phosphorylation of CFBP. They co-localize in actinenriched structures, and overexpression of CFBP induced morphological changes with actin reorganization. Furthermore, CFBP accelerated the EGF receptor's down-regulation by facilitating the recruitment of Cbl to the CD2AP/CIN85 complex. Two spliced variants of CFBP lacking either exon 5 or 8 are also expressed, and the variant lacking exon 5 without the proline-rich motif lacks the ability to bind to the CIN85/CD2AP family. The CFBP protein seems to play a key role in the ligand-mediated internalization and down-regulation of the EGF receptor.

Suppression of the ligand-mediated down-regulation of epidermal growth factor receptor by Ymer, a novel tyrosine-phosphorylated and ubiquitinated protein.

The ligand-mediated down-regulation of the growth factor receptors is preceded by the involvement of various other factors. In particular, a ubiquitin ligase, Cbl, plays a central role in this event. Several candidates that have potential effects on the negative control of the epidermal growth factor (EGF) receptor have now been identified by our recent studies in phospho-proteomics. Among these molecules, we focus on characterizing a novel protein, Ymer, which is a tyrosine-phosphorylated and ubiquitinated protein. Ymer is found to be phosphorylated at tyrosine 145 and 146 upon EGF stimulation, and lysine 129 of Ymer has been identified as a ubiquitination site. Ymer has two motifs interacting with the ubiquitin (MIU) domains that might function as a binding site for the ubiquitinated EGF receptor. Although Ymer and EGF receptors are associated in an EGF-dependent manner, their interaction is required not only for MIU domains but also for the tyrosine phosphorylation of Ymer. Phosphorylated Ymer is mainly located at the plasma membrane with EGF receptor and functions in its endocytosis and degradation. Furthermore, EGF-mediated secondary modifications of an activated-EGF receptor are inhibited by overexpressing Ymer in COS7 cells. Therefore, Ymer may have competitive effects on the activation of the EGF receptor. Our findings suggest that Ymer functions as a novel inhibitor for the down-regulation of the EGF receptor and plays a crucial role for regulating the amount of the EGF receptor on the cell surface membrane.

Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A.

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin.

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.

Phosphorylation of microtubule-associated protein tau by Ca2+/calmodulin-dependent protein kinase II in its tubulin binding sites.

The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain.

Regulation of exocytosis through Ca2+/ATP-dependent binding of autophosphorylated Ca2+/calmodulin-activated protein kinase II to syntaxin 1A.

Syntaxin 1A/HPC-1 is a key component of the exocytotic molecular machinery, namely, the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor mechanism. Although >10 syntaxin-binding proteins have been identified, they cannot completely explain the regulation of exocytosis. Thus, novel proteins may interact with syntaxin. Because exocytosis requires both Ca2+ and ATP, we searched for Ca2+/ATP-dependent syntaxin-binding proteins from the rat brain and discovered Ca2+/calmodulin-activated protein kinase II (CaMKII)-alpha. At Ca2+ concentrations of >10(-6) m, only autophosphorylated CaMKII bound to syntaxin. Bound CaMKII was released from syntaxin by EGTA or by phosphatase, indicating that the binding is reversible. CaMKII bound to the linker domain of syntaxin, unlike any other known syntaxin-binding proteins. CaMKII-syntaxin complexes were also detected in synaptosomes by immunoprecipitation, and when reconstituted in vitro, they recruited larger amounts of synaptotagmin and SNAP-25 than syntaxin alone. The microinjected CaMKII-binding domain of syntaxin specifically affected exocytosis in chromaffin cells and in neurons. These results indicate that the Ca2+/ATP-dependent binding of CaMKII to syntaxin is an important process in the regulation of exocytosis.