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Beta-tubulin binds Src homology 2 domains through a region different from the tyrosine-phosphorylated
1Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan.
Abstract:
Src homology 2 (SH2) domains have been demonstrated to bind tyrosine-phosphorylated proteins that participate in signaling by growth factors and oncogenes by recognizing amino acid sequences containing phosphotyrosine residue. We found that SH2 domains such as Ash/Grb2, the 85-kDa subunit of phosphatidylinositol 3-kinase, and phospholipase Cgamma1 also bind beta-tubulin through a different region that recognizes phosphotyrosine in vitro and in vivo. Furthermore, binding occurs even when the SH2 domain is occupied by tyrosine-phosphorylated epidermal growth factor receptors. Using deleted constructs of Ash/Grb2 SH2, we found that carboxyl-terminal beta strands E and F, and alpha helix B (region "c") are required for binding. A synthetic peptide (FLWVVKFNSLNELVDYH) composed of region c inhibited the binding of beta-tubulin to the SH2 domains of Ash/Grb2, phosphatidylinositol 3-kinase, and phospholipase Cgamma1. The co-localization of SH2 proteins and microtubules is also confirmed by immunostaining. These data suggest that microtubules play important roles in the assembly of signaling molecules complexes containing SH2 proteins.
Insights
Src homology 2 (SH2) domains bind beta-tubulin via a unique region, independent of phosphotyrosine binding. This interaction is crucial for assembling signaling complexes involving SH2 proteins and microtubules.
Area of Science:
- Cellular signaling
- Molecular biology
- Protein-protein interactions
Background:
- Src homology 2 (SH2) domains recognize phosphotyrosine residues on proteins, mediating signaling pathways for growth factors and oncogenes.
- SH2 domains are critical for signal transduction by growth factors and oncogenes.
Purpose of the Study:
- To investigate novel binding interactions of SH2 domains beyond canonical phosphotyrosine recognition.
- To explore the role of beta-tubulin in SH2 domain-mediated signaling complex assembly.
Main Methods:
- In vitro and in vivo binding assays using SH2 domain constructs and beta-tubulin.
- Analysis of SH2 domain deletion mutants to identify binding regions.
- Peptide inhibition assays to confirm binding specificity.
- Immunostaining to assess co-localization of SH2 proteins and microtubules.
Main Results:
- SH2 domains, including those in Ash/Grb2, phosphatidylinositol 3-kinase, and phospholipase Cgamma1, bind beta-tubulin through a distinct region.
- This binding occurs independently of phosphotyrosine binding and even when the SH2 domain is occupied.
- Specific beta-tubulin binding requires carboxyl-terminal beta strands E and F, and alpha helix B of the SH2 domain.
- A synthetic peptide mimicking this region inhibited beta-tubulin binding to multiple SH2 domains.
- SH2 proteins and microtubules were observed to co-localize within cells.
Conclusions:
- Microtubules are integral to the formation of signaling complexes containing SH2 proteins.
- SH2 domains possess a previously unrecognized interaction site for beta-tubulin, independent of phosphotyrosine.
- This interaction suggests a novel mechanism for scaffolding signaling molecules via the microtubule network.