G3BP (Ras-GTPase-activating protein SH3 domain binding protein), a protein which binds to RasGAP SH3 domain, belongs to RNA-binding protein family, implicating in the downstream of Ras signaling. G3BP harbors the activities of endoribonuclease and DNA helicase, and can induce stress granules formation. G3BP plays a general role in the signal pathways of cell proliferation, differentiation, apoptosis and RNA metabolism. It has been shown to be over-expressed in a number of human malignancies and has a close relationship with tumor invasion and metastasis. Given that it has been implicated in several pathways that are known to be involved in cancer biology, G3BP may provide a new target for cancer therapy.
Animals ; Carrier Proteins ; genetics ; metabolism ; DNA Helicases ; Drug Delivery Systems ; GTPase-Activating Proteins ; therapeutic use ; Humans ; Molecular Sequence Data ; Neoplasms ; drug therapy ; metabolism ; pathology ; Peptide Fragments ; therapeutic use ; Phosphorylation ; Poly-ADP-Ribose Binding Proteins ; RNA Helicases ; RNA Recognition Motif Proteins ; Signal Transduction ; ras GTPase-Activating Proteins ; metabolism ; src Homology Domains ; genetics

OBJECTIVETo study the interaction between ShcD and TrkC and to reveal the molecular mechanism of the downstream signal transduction of TrkC.

METHODSYeast two-hybrid assay was used. The intracellular domains of TrkC and TrkC mutants were cloned into pAS2-1, and ShcD and its four domains (CH2, PTB, CH1, and SH2 domains) were cloned into pACT2 vector respectively. The constructs were separately cotransformed into yeast. beta-galactosidase activity was measured to detect their interactions. TrkC was cloned into pmRFP (carrying red fluorescent protein), and ShcD was cloned into pEGFP (carrying green fluorescent protein). pmRFP-TrkC and pEGFP-ShcD were co-transfected into 293T cells, and then the cells were fixed and subjected to confocal analysis to study their subcellular localization.

RESULTSShcD interacted with TrkC but not with kinase dead mutant TrkCM1(K572A). Both PTB and SH2 domains were capable of binding to TrkC, and PTB domain bound NPQY motif of TrkC. ShcD colocalized with TrkC throughout the cytoplasm and in the plasma membrane in 293T cells.

CONCLUSIONShcD binds to TrkC in a kinase-activity-dependent manner through its PTB and SH2 domains.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Binding Sites ; Cells, Cultured ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Protein Binding ; Receptor, trkC ; genetics ; metabolism ; Shc Signaling Adaptor Proteins ; genetics ; metabolism ; Transfection ; Transformation, Bacterial ; Two-Hybrid System Techniques ; src Homology Domains ; genetics

OBJECTIVE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarizationactivated currents (Ih) that participate in regulating neuronal membrane potential and contribute critically to pacemaker activity, promoting synchronization of neuronal networks. However, distinct regional and cellular localization of HCN channels in the brain have not been precisely defined. Aim of this study was to verify the precise cellular location of HCN1 channels in rat cerebellum to better understand the physiological role these channels play in synaptic transmission between CNS neurons. METHODS: HCN1 expression in rat brain was analyzed using immunohistochemistry and electron-microscopic observations. Postsynaptic density-95 (PSD-95), otherwise known as locating and clustering protein, was also examined to clarify its role in the subcellular location of HCN1 channels. In addition, to presume the binding of HCN1 channels with PSD-95, putative binding motifs in these channels were investigated using softwaresearching method. RESULTS: HCN1 channels were locally distributed at the presynaptic terminal of basket cell and exactly corresponded with the location of PSD-95. Moreover, nine putative SH3 domain of PSD-95 binding motifs were discovered in HCN1 channels from motif analysis. CONCLUSION: Distinct localization of HCN1 channels in rat cerebellum is possible, especially when analyzed in conjunction with the SH3 domain of PSD-95. Considering that HCN1 channels contribute to spontaneous rhythmic action potentials, it is suggested that HCN1 channels located at the presynaptic terminal of neurons may play an important role in synaptic plasticity.
Action Potentials ; Animals ; Brain ; Cerebellar Cortex* ; Cerebellum ; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels ; Immunohistochemistry ; Membrane Potentials ; Neurons ; Plastics ; Presynaptic Terminals ; Rats* ; src Homology Domains ; Synaptic Transmission

BACKGROUNDLaryngeal carcinoma is a common malignant tumor of the upper respiratory tract, and in 95% of cases the tumor is laryngeal squamous cell carcinoma (LSCC). The abnormity of SH3-domain GRB2-like 2 (SH3GL2) gene was found in LSCC. In order to clarify the relationship between SH3GL2 gene and LSCC, we evaluated the expression of the SH3GL2 gene in LSCC.

METHODReal-time PCR, immunohistochemistry and Western blotting were used to detect the mRNA and protein expression and find the various rules of SH3GL2 gene in LSCC.

RESULTSThe result of real-time PCR showed that the expression level of SH3GL2 mRNA in LSCC tissue was apparently down-regulated; immunohistochemical analysis showed that SH3GL2 protein was mainly located in cytoplasm, the rate of positive cells and SH3GL2 protein expression level were fluctuated with the pathological classification of LSCC; the result of Western blotting showed that SH3GL2 protein was down-regulated significantly in LSCC samples, especially in metastatic lymph nodes.

CONCLUSIONSThese results suggest that SH3GL2 is a LSCC related gene and its expression level is fluctuated with the pathological classification which indicate that SH3GL2 participates in the development and progression of LSCC. And it may be considered as a novel tumor marker to find both a new anti-oncogene and relative factors of invasion and metastasis of laryngeal carcinoma.

Adaptor Proteins, Signal Transducing ; analysis ; genetics ; Blotting, Western ; Carcinoma, Squamous Cell ; chemistry ; genetics ; Humans ; Immunohistochemistry ; Laryngeal Neoplasms ; chemistry ; genetics ; Polymerase Chain Reaction ; src Homology Domains

The c-Abl nonreceptor tyrosine kinase is activated in the cellular responses to genotoxic, oxidative and other forms of stress. Using tagged forms of c-Abl, the present studies demonstrate that c-Abl forms homodimers in cells. The results show that the c-Abl N-terminal regions interact with the corresponding C-terminal regions of both partners in the dimmer. Specifically, the c-Abl SH3 domain binds to a proline-rich motif at amino acids 958-982 in the c-Abl C-terminal region. Deletion of the proline-rich motif disrupts dimmer formation. These findings provide the first evidence that c-Abl forms homodimers and indicate that homodimerization can contribute to the regulation of c-Abl activity.
Humans ; Protein Multimerization ; Proto-Oncogene Proteins c-abl ; genetics ; metabolism ; src Homology Domains

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.
Adaptor Proteins, Signal Transducing ; Amino Acid Sequence ; Animals ; COS Cells/enzymology ; Cercopithecus aethiops ; Enzyme Activation ; Epidermal Growth Factor/*pharmacology ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate/metabolism ; Molecular Sequence Data ; Phospholipase C/chemistry/*metabolism ; Phosphoric Monoester Hydrolases/chemistry/*metabolism ; Protein Binding ; Signal Transduction ; src Homology Domains/*physiology

Human mesenchymal stem cells (MSC) are one kind of adult stem cells that can self-renew and give rise to one or more mesenchymal tissues, existing in bone marrow and other tissues. Not similar to CD34 recognizing hematopoietic stem cells, no such marker can be used yet to identify MSC. To isolate and identify MSC from bone marrow, anti-SH2 and SH3 monoclonal antibodies as markers to identify MSC were used. Two monoclonal antibodies were purified from ascites of SH2 and SH3 hybridomas-inoculated mice, flow cytometry and immunohistochemistry were used to identify plastic-adherent cultured MSC. And SH2 and SH3 antigen positive cells were isolated from bone marrow mononuclear cells (BMMNC) by immunobeads covered with secondary antibodies. And anti-SH2 and CD105 McAbs were used to label MSC at the same time to clarify whether they recognize the same antigen. The results showed that about 80% of MSC were antigens SH3 and SH2 positive. The SH2 and SH3 positive-selected cells were MSC while MSC accounted for less than 1% of negative-selected cells. When cells were labeled by SH2 McAb, they could not be labeled by CD105 simutaneously. In conclusion, antigens SH2 and SH3 are specific markers to identify and isolate MSC. Anti-SH2 McAb can replace anti-CD105 McAb to identify the specific marker on MSC.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Bone Marrow Cells ; cytology ; immunology ; Cells, Cultured ; Flow Cytometry ; Humans ; Immunohistochemistry ; Immunomagnetic Separation ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; Mice ; src Homology Domains ; immunology

0.7). The "RUNX-1" was increased its expression in 2 hours group and "RUN and SH3 domain containing 1" was increased its expression in 4 hours group. The "CC020415", "cyclin L1", "interferon regulatory factor1", "early growth response 1", "immediate early response 2", and "immediate early response 3" genes were increased their expression in 2 and 4 hours after FISS application. In conclusion, we could find many genes that were probably related to the FISS application. Interestingly, most of them were placed in similar molecular pathways and these findings improve the reliability of chip data and usefulness in overall screening. From this experiment, we could find many items for further study and it will make improvement in the understanding of intracellular events in response to FISS.]]>
DNA, Complementary ; Fibroblasts ; Gene Expression ; Homeostasis ; Humans ; Mass Screening ; Mouth ; Mouth Mucosa ; Oligonucleotide Array Sequence Analysis ; RNA ; src Homology Domains

Domain database is essential for domain property research. Eliminating redundant information in database query is very important for database quality. Here we report the manual construction of a non-redundant human SH2 domain database. There are 119 human SH2 domains in 110 SH2-containing proteins. Human SH2s were aligned with ClustalX, and a homologous tree was generated. In this tree, proteins with similar known function were classified into the same group. Some proteins in the same group have been reported to have similar binding motifs experimentally. The tree might provide clues about possible functions of hypothetical proteins for further experimental verification.
Amino Acid Sequence ; Computational Biology ; Databases, Protein ; Humans ; Molecular Sequence Data ; Sequence Alignment ; src Homology Domains ; genetics

In this study, the molecular mechanism of tyrosine phosphorylation of Roundabout (Robo), the transmembrane receptor for slits, was investigated. The tyrosine phosphorylation of intracellular portion of Robo was increased by the treatment of tyrosine phosphatase inhibitors in human embryonic kidney cells transfected with Robo. The Robo tyrosine phosphorylation was inhibited by the treatment of Src family kinase inhibitor, PP2. The co-transfection of constitutively active form of Fyn, not the dominant negative form of Fyn, and Robo dramatically enhanced the tyrosine phosphorylation of Robo. Furthermore, the SH2 domain of Fyn, which binds to phosphorylated tyrosine residues, interact with Robo, and the interaction was increased by the inhibition of tyrosine phosphatases. These findings indicate that the tyrosine phosphorylation of Robo is regulated by Fyn.
Humans ; Kidney ; Phosphoric Monoester Hydrolases ; Phosphorylation* ; Phosphotransferases ; src Homology Domains ; Tyrosine*