Modified SH2 domain to phototrap and identify phosphotyrosine proteins from subcellular sites within cells

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.protein phosphorylation | proteomics | signal transduction

Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation

“…Studies using purified insulin receptor and Ras GTPases have suggested that Ras proteins may be phosphorylated on Tyr residues (37), and a recent report using the modified SH2 domain to phototrap potential Tyr residues identified Y32 and Y64 as possible sites of phosphorylation on H-Ras in vitro (27). However, the kinase responsible for and the functional significance of Ras phosphorylation remained unknown.…”

“…Y32, Y64, Y96, and Y157 were phosphorylated by c-Src in vitro. Interestingly, a peptide array demonstrated that phosphorylated H-Ras peptides can bind to the GST-c-Abl SH2 domain, with Y32-containing peptide being the most robust (27). We next asked which N-Ras sites, if any, were phosphorylated by c-Src in vivo.…”

Phosphorylation of C-terminal tyrosine 526 in FUS impairs its nuclear import

“…As tyrosine residues can undergo post-translational modifications, and phenylalanine and glutamate residues might serve as mimetics of unphosphorylated and phosphorylated tyrosine, respectively, we also generated Y526F and Y526E constructs (Derkinderen et al, 2005;Uezu et al, 2012). In transiently transfected HeLa cells (Fig.…”

The Genesis of Tyrosine Phosphorylation

“…Many nonreceptor TKs interact with targets via their SH2 or SH3 domains, and a new method for identifying SH2 and SH3 domain binding partners in the cell has recently been developed. This method takes advantage of "unnatural amino acid" (UAA) technology, in which a UV photoactivatable UAA is incorporated into a protein domain at specific sites on its ligand-interacting surface in appropriately engineered cells, allowing cross-linking to bound proteins when cells are irradiated with UV, and recovery of the tagged domains for MS analysis (Okada et al 2011;Uezu et al 2012). …”