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Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells
Akiyoshi Uezu1, Hirokazu Okada, Hideji Murakoshi
1Department of Cell Biology and Howard Hughes Medical Institute and Neurobiology Department, Duke University Medical School, Durham, NC 27710, USA.
Abstract:
Spatial regulation of tyrosine phosphorylation is important for many aspects of cell biology. However, phosphotyrosine accounts for less than 1% of all phosphorylated substrates, and it is typically a very transient event in vivo. These factors complicate the identification of key tyrosine kinase substrates, especially in the context of their extraordinary spatial organization. Here, we describe an approach to identify tyrosine kinase substrates based on their subcellular distribution from within cells. This method uses an unnatural amino acid-modified Src homology 2 (SH2) domain that is expressed within cells and can covalently trap phosphotyrosine proteins on exposure to light. This SH2 domain-based photoprobe was targeted to cellular structures, such as the actin cytoskeleton, mitochondria, and cellular membranes, to capture tyrosine kinase substrates unique to each cellular region. We demonstrate that RhoA, one of the proteins associated with actin, can be phosphorylated on two tyrosine residues within the switch regions, suggesting that phosphorylation of these residues might modulate RhoA signaling to the actin cytoskeleton. We conclude that expression of SH2 domains within cellular compartments that are capable of covalent phototrapping can reveal the spatial organization of tyrosine kinase substrates that are likely to be important for the regulation of subcellular structures.
Insights
Researchers developed a new method to identify tyrosine kinase substrates using a light-activated unnatural amino acid-modified Src homology 2 (SH2) domain. This technique captures phosphotyrosine proteins within specific cellular compartments, revealing their spatial organization and regulation.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Spatial regulation of tyrosine phosphorylation is crucial for cellular functions.
- Identifying transient and spatially organized phosphotyrosine substrates is challenging.
- Tyrosine kinases play key roles in cell signaling and regulation.
Purpose of the Study:
- To develop a novel method for identifying tyrosine kinase substrates based on their subcellular localization.
- To investigate the spatial organization of tyrosine kinase substrates within specific cellular compartments.
- To capture and characterize transient phosphotyrosine proteins in vivo.
Main Methods:
- Utilized an unnatural amino acid-modified Src homology 2 (SH2) domain as a photoprobe.
- Engineered the SH2 domain to covalently trap phosphotyrosine proteins upon light exposure.
- Targeted the SH2 photoprobe to specific cellular structures like the actin cytoskeleton, mitochondria, and membranes.
Main Results:
- Successfully captured tyrosine kinase substrates unique to different cellular regions.
- Demonstrated the phosphorylation of RhoA on two tyrosine residues within its switch regions.
- Provided evidence that localized phosphorylation may modulate RhoA signaling to the actin cytoskeleton.
Conclusions:
- Expression of engineered SH2 domains within cellular compartments enables covalent phototrapping of tyrosine kinase substrates.
- This approach reveals the spatial organization of phosphotyrosine proteins critical for subcellular structure regulation.
- The method offers new insights into the spatial control of signaling pathways mediated by tyrosine kinases.
