Shb is a ubiquitously expressed Src homology 2 protein
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Published on: October 19, 2014
1Department of Medical Cell Biology, Uppsala University, Sweden.
Abstract:
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.
Insights
Researchers identified Shb, a novel adaptor protein with Src homology 2 (SH2) and Src homology 3 (SH3) binding domains. Shb is involved in signal transduction pathways activated by growth factors like platelet-derived growth factor (PDGF).
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Identification of serum-inducible genes in insulin-producing beta TC-1 cells is crucial for understanding cellular regulation.
- Serum stimulation can activate complex signaling pathways involving growth factors and tyrosine kinase receptors.
Purpose of the Study:
- To identify serum-inducible genes in the beta TC-1 cell line.
- To characterize a novel gene, Shb, identified through subtraction screening.
- To investigate the potential role of Shb in signal transduction pathways.
Main Methods:
- Library subtraction screening of serum-deprived and serum-restimulated beta TC-1 cells.
- cDNA cloning and sequence analysis to identify novel genes.
- Western blot analysis to detect protein expression in various tissues.
- Transient and stable transfection of Shb cDNA in cell lines (COS-1, pig aortic endothelial cells).
- Fusion protein analysis to study protein-ligand interactions.
Main Results:
- A novel cDNA, Shb, was identified, encoding a protein with Src homology 2 (SH2) and proline-rich domains (potential Src homology 3 (SH3) binding sites).
- Shb mRNA (3.1 kb) is expressed in multiple mouse tissues and cell lines.
- Western blot detected Shb proteins of 67, 56, and 40 kDa in various tissues, with increased expression upon transfection.
- Shb's SH2 domain specifically bound to autophosphorylated platelet-derived growth factor (PDGF) beta-receptor, but not the epidermal growth factor (EGF) receptor.
Conclusions:
- Shb is a novel SH2-containing adaptor protein with proline-rich domains.
- Shb is likely involved in signal transduction pathways mediated by ligand-activated tyrosine kinase receptors, particularly the PDGF receptor.
- The findings suggest Shb plays a role in cellular responses to growth factor stimulation.
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