Adapter protein which regulates several signal transduction cascades by linking activated receptors to downstream signaling components. May play a role in angiogenesis by regulating FGFR1, VEGFR2 and PDGFR signaling. May also play a role in T-cell antigen receptor/TCR signaling, interleukin-2 signaling, apoptosis and neuronal cells differentiation by mediating basic-FGF and NGF-induced signaling cascades. May also regulate IRS1 and IRS2 signaling in insulin-producing cells.
The mechanisms leading to focal adhesion kinase (FAK) activation remain obscure. We have investigated the role of the adaptor protein Shb in cell spreading and the regulation of FAK phosphorylation in immortalised brain endothelial (IBE) cells. Fibroblast growth factor 2 (FGF-2) stimulation lead to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding (PTB) domain of Shb. IBE cells overexpressing wild-type or R522K Shb (with an inactive Src homology 2 (SH2) domain) displayed increased FAK phosphorylation as well as enhanced spreading when seeded on collagen. FGF-2-induced tyrosine phosphorylation of Shb was dependent upon Src activity but independent of FAK activation. The use of Rat-1 fibroblasts overexpressing a temperature sensitive v-Src (tsLA29) confirmed that active Src enhanced Shb phosphorylation. The data indicate that Shb binds directly to FAK and regulates its phosphorylation leading to enhanced cell spreading in a Src-dependent manner.
Eur. J. Biochem. 269, 3279-3288 (2002)[PubMed:12084069]
This study addresses the interactions between the adaptor protein Shb and components involved in T cell signalling, including SLP-76, Gads, Vav and ZAP70. We show that both SLP-76 and ZAP70 co-immunoprecipitate with Shb in Jurkat T cells and that Shb and Vav co-immunoprecipitate when cotransfected in COS cells. We also demonstrate, utilizing fusion protein constructs, that SLP-76, Gads and Vav associate independently of each other to different domains or regions, of Shb. Overexpression of an SH2 domain-defective Shb causes diminished phosphorylation of SLP-76 and Vav and consequently decreased activation of c-Jun kinase upon T cell receptor (TCR) stimulation. Shb was also found to localize to glycolipid-enriched membrane microdomains (GEMs), also called lipid rafts, after TCR stimulation. Our results indicate that upon TCR stimulation, Shb is targeted to these lipid rafts where Shb aids in recruiting the SLP-76-Gads-Vav complex to the T cell receptor zeta-chain and ZAP70.
The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-beta, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-beta were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-beta signals.
The Src homology (SH) 2 domain adaptor protein Shb has previously been shown to interact with the platelet-derived growth factor (PDGF)-beta receptor. In this study we show an association between Shb and the PDGF-alpha receptor which is mediated by the SH2 domain of Shb and involves tyrosine residue 720 in the kinase insert domain of the receptor. To assess the role of Shb in PDGF-mediated signaling, we have overexpressed wild-type Shb or Shb carrying a mutation (R522K) which renders the SH2 domain inactive, in Patch mouse (PhB) fibroblasts expressing both PDGF receptors (PhB/Ralpha). Overexpression of wild-type Shb, but not the R522K Shb mutant, affected PDGF-mediated reorganization of the cytoskeleton by decreasing membrane ruffle formation and stimulating the generation of filopodia relative the parental control cells. In addition, the PDGF-induced receptor-associated phosphatidylinositol 3'-kinase activity and phosphorylation of Akt was similar in both PhB/Ralpha/Shb and PhB/Ralpha/ShbR522K cells compared with the parental control, whereas the activation of Rac in response to PDGF-BB was diminished only in the PhB/Ralpha/Shb cells. We conclude that Shb plays a role in PDGF-dependent regulation of certain cytoskeletal changes by modulating the ability of PDGF to activate Rac.
To assess a possible role for the Src homology 2 (SH2) domain adaptor protein Shb in PC12 cell signal transduction and differentiation, we have investigated the expression of Shb in PC12 cells and found that the differentiation factors nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), as well as the PC12 cell mitogen epidermal growth factor, increased Shb protein expression and Shb mRNA steady-state levels. Two PC12 cell clones stably overexpressing the Shb cDNA exhibited enhanced NGF- or bFGF-induced differentiation, assessed as neurite outgrowth. This effect showed no direct correlation to mitogen-activated protein kinase activation, although the mitogen-activated protein kinase/kinase inhibitor PD-98059 caused a partial inhibition of neurite outgrowth. Furthermore, it was found that the Shb-overexpressing cells extended neurites in response to epidermal growth factor. The effects of Shb overexpression on neurite outgrowth required a functional SH2 domain because PC12 cells expressing Shb with an inactivated SH2 domain did not differentiate more readily in response to NGF. Tyrosine phosphorylation of the p13 Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target protein in response to bFGF was strongly inhibited by Shb overexpression, without correlating with the corresponding rate of neurite outgrowth. NGF-induced tyrosine phosphorylation of phospholipase Cgamma, TrkA, and Shc was unaltered in the Shb-overexpressing cells, whereas that of Shb was greatly enhanced in these cells compared with control PC12-neo cells. In addition, an NGF-activated Mr 140,000 phosphotyrosine protein was found to be associated with Shb in immunoprecipitation experiments. The data in this study suggest that Shb is involved in transmission of NGF- and bFGF-dependent differentiation signals in PC12 cells.
Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells.
To understand the role of the Src homology 2 (SH2) domain protein Shb in the signal transduction of tyrosine kinase receptor, NIH3T3 cells were transfected with a DNA construct expressing the Shb cDNA (NIHSHB cells). The NIHSHB cells expressed elevated levels of proteins with the estimated molecular weights of 77, 66 and 55 kDa as determined by immunoblotting. In contrast to the control cells, the NIHSHB cells failed to increase in cell number in the presence of 1% serum. This effect was largely due to apoptosis, since staining of pyknotic nuclei was observed using the terminal transferase labeling method. The NIHSHB cells displayed similar levels of c-myc mRNA and decreased contents of the p53 protein after culture in 1% serum compared with control cells. The addition of platelet-derived growth factor (PDGF-BB) restored the growth of the NIHSHB cells, whereas insulin-like growth factor-1 (IGF-1) failed to affect the proliferation of Shb overexpressing cells in 1% serum. We conclude that Shb overexpression is associated with cell degeneration under certain conditions, and that Shb could transduce apoptotic signals from tyrosine kinase receptors.
Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.
Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGF-induced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.
BACKGROUND: Overexpression of the Src homology 2 domain protein Shb in beta-cells of transgenic mice has been shown to promote an increased beta-cell mass. To investigate the mechanisms by which Shb controls the beta-cell mass, we have presently studied the effects of Shb overexpression on the IRS-1-induced signaling pathway in mouse islet beta-cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns. MATERIALS AND METHODS: Shb overexpression was achieved in RINm5F cells by selection of stable clones or by FACS purification of transiently transfected cells. For Shb overexpression in primary mouse islet cells, a Shb-transgene mouse was used. Cell proliferation and death rates were determined using flow cytometry. Serum-, insulin-, and IGF-1-stimulated signaling events were studied by immunoblot, immunoprecipitation, and in vitro kinase procedures. RESULTS: Transient Shb overexpression in RINm5F cells resulted in increased proliferation. Both Shb-overexpressing RINm5F cells and islet cells from transgenic mice (islet Shb) exhibited increased basal tyrosine phosphorylation of IRS-1. Shb overexpression resulted also in the assembly and activation of a multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK, and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpressing cells. Finally, Shb overexpression did not affect insulin-induced phosphorylation of the PI3K-antagonist PTEN. CONCLUSION: It is concluded that the Shb-induced alterations in the IRS-1/PI3K/Akt pathway may be relevant to the understanding of growth and death patterns of insulin-producing cells.
Interacting selectively and non-covalently and simultaneously with one or more signal transduction molecules, usually acting as a scaffold to bring these molecules into close proximity either using their own SH2/SH3 domains (e.g. Grb2) or those of their target molecules (e.g. SAM68).
To identify serum-inducible genes in the insulin-producing cell line beta TC-1, a library subtraction screening procedure was performed on serum-deprived (G0) and serum-restimulated (G1) insulin-producing beta TC-1 cells. A cDNA containing a motif with strong homology to Src homology 2 (SH2) domains was found using this procedure and called Shb. The Shb cDNA contains two methionine codons in its N-terminus and thus may code for two proteins of 67 and 56 kDa, each with one SH2 domain in its C-terminus. No other structural similarity to proteins with catalytic activity could be detected, suggesting that Shb is a so called adaptor. Shb contains the proline-rich sequence PPPGPGR between the two proposed initiator methionines which resembles a sequence for binding to Src homology 3 (SH3) domains. A second proline-rich sequence was detected after the second methionine codon. The Shb cDNA hybridized to a similar or identical mRNA of 3.1 kb expressed in mouse brain, liver, kidney, heart, NIH3T3 fibroblasts and beta TC-1 cells. Western blot analysis of the same tissues using an antiserum directed against a synthetic peptide corresponding to a part of the SH2 domain of Shb, revealed reactivity with two proteins of 56 and 67 kDa. In addition, a third reactive component of 40 kDa was detected in most tissues. Transfection and transient expression of the Shb cDNA in COS-1 cells yielded increased expression of the 67, 56 and 40 kDa proteins. Transfection and stable expression of the Shb cDNA in pig aortic endothelial cells showed increased expression primarily of the 67 kDa protein. A fusion protein consisting of the SH2 domain of Shb linked to glutathione S-transferase showed increased binding to glycoproteins of cells stimulated with platelet-derived growth factor (PDGF-BB). Furthermore, the autophosphorylated PDGF beta-receptor but not the autophosphorylated epidermal growth factor (EGF) receptor bound specifically to immobilized fusion protein. It is concluded that Shb is a novel SH2-containing protein with proline-rich domains and therefore probably involved in the signal-transduction of some ligand-activated tyrosine kinase receptors.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
The process in which relatively unspecialized cells, e.g. embryonic or regenerative cells, acquire specialized structural and/or functional features that characterize the cells, tissues, or organs of the mature organism or some other relatively stable phase of the organism's life history. Differentiation includes the processes involved in commitment of a cell to a specific fate and its subsequent development to the mature state.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
To identify serum-inducible genes in the insulin-producing cell line beta TC-1, a library subtraction screening procedure was performed on serum-deprived (G0) and serum-restimulated (G1) insulin-producing beta TC-1 cells. A cDNA containing a motif with strong homology to Src homology 2 (SH2) domains was found using this procedure and called Shb. The Shb cDNA contains two methionine codons in its N-terminus and thus may code for two proteins of 67 and 56 kDa, each with one SH2 domain in its C-terminus. No other structural similarity to proteins with catalytic activity could be detected, suggesting that Shb is a so called adaptor. Shb contains the proline-rich sequence PPPGPGR between the two proposed initiator methionines which resembles a sequence for binding to Src homology 3 (SH3) domains. A second proline-rich sequence was detected after the second methionine codon. The Shb cDNA hybridized to a similar or identical mRNA of 3.1 kb expressed in mouse brain, liver, kidney, heart, NIH3T3 fibroblasts and beta TC-1 cells. Western blot analysis of the same tissues using an antiserum directed against a synthetic peptide corresponding to a part of the SH2 domain of Shb, revealed reactivity with two proteins of 56 and 67 kDa. In addition, a third reactive component of 40 kDa was detected in most tissues. Transfection and transient expression of the Shb cDNA in COS-1 cells yielded increased expression of the 67, 56 and 40 kDa proteins. Transfection and stable expression of the Shb cDNA in pig aortic endothelial cells showed increased expression primarily of the 67 kDa protein. A fusion protein consisting of the SH2 domain of Shb linked to glutathione S-transferase showed increased binding to glycoproteins of cells stimulated with platelet-derived growth factor (PDGF-BB). Furthermore, the autophosphorylated PDGF beta-receptor but not the autophosphorylated epidermal growth factor (EGF) receptor bound specifically to immobilized fusion protein. It is concluded that Shb is a novel SH2-containing protein with proline-rich domains and therefore probably involved in the signal-transduction of some ligand-activated tyrosine kinase receptors.
Protein involved in angiogenesis, the sprouting or splitting of capillaries from pre-existing vasculature. Angiogenesis plays an important role for example during embryonic development, normal growth of tissues and maintenance of the normal vasculature, wound healing, tumor growth and metastasis.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in differentiation, the developmental process of a multicellular organism by which cells become specialized for particular functions. Differentiation requires selective expression of the genome; the fully differentiated state may be preceded by a stage in which the cell is already programmed for differentiation but is not yet expressing the characteristic phenotype determination. Also used for fungal conidiation proteins, and for some bacteria that present specialization of function in cell types, such as Caulobacter crescentus.
Protein involved in development, the process whereby a multicellular organism develops from its early immature forms, e.g., zygote, larva, embryo, into an adult.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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