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Src homology region 2 domains direct protein-protein interactions in signal transduction
M F Moran1, C A Koch, D Anderson
1Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
Abstract:
Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.
Insights
The study shows that Src homology region 2 (SH2) domains mediate interactions between signaling proteins and phosphotyrosine ligands like the EGF receptor. These SH2 domains act as autonomous units, directing protein binding and regulating growth factor signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Interactions
Background:
- Cytoplasmic proteins regulating signal transduction and cellular transformation often contain conserved domains.
- Src homology region 2 (SH2) domains are found in proteins like p21ras GTPase-activating protein (GAP) and oncoproteins.
Purpose of the Study:
- To provide direct evidence that SH2 domains mediate interactions between signaling proteins and phosphotyrosine ligands.
- To investigate the role of SH2 domains in protein complex formation and signal transduction.
Main Methods:
- In vitro reconstitution of protein complexes using bacterial polypeptides containing SH2 domains.
- Analysis of protein-protein interactions in src-transformed and EGF-stimulated cells.
- Use of fusion proteins containing SH2 domains to study binding affinities.
Main Results:
- SH2 domains mediate interactions between diverse signaling proteins (e.g., GAP) and phosphotyrosine ligands (e.g., EGF receptor).
- The N-terminal GAP SH2 domain specifically reconstitutes the association between GAP and a 62-kDa phosphotyrosine protein (p62) in vitro.
- SH2 domain-dependent phosphorylation of p62 by tyrosine kinases correlates with cellular transforming activity.
- SH2 domains function as autonomous units, directing signaling proteins to bind specific phosphotyrosine-containing polypeptides.
Conclusions:
- SH2 domains are crucial for the specific binding of signaling proteins to phosphotyrosine-containing molecules.
- These interactions are essential for regulating intracellular signaling pathways activated by growth factors.
- SH2 domains play a key role in mediating signal transduction and cellular transformation.