Caged Thiophosphotyrosine Peptides

Abstract

Caged but not trapped! Caged reagents are molecules from which biological effectors are released by photolysis. Here, p‐hydroxyphenacyl bromide is used to derivatize a model peptide containing a thiophosphotyrosine residue in order to mimic phosphorylation at tyrosine. Upon photolysis, a biologically active peptide is released. Peptides and proteins caged on thiophosphotyrosine will be useful in signal transduction research.

Auto-thiophosphorylation activity of Src tyrosine kinase

“…In our previous studies [16], we showed that the Src family kinase Hck can catalyze thiophosphorylation of a peptide substrate in the presence of CoCl 2 . In those studies, an endpoint assay was used; we analyzed aliquots from reactions by analytical HPLC, and confirmed peptide thiophosphorylation by mass spectrometry.…”

“…We and others have previously observed that the inclusion of thiophilic divalent cations such as Co 2+ or Ni 2+ enhances the thiophosphorylation activity of protein kinases [10, 16]. Inclusion of Mn 2+ together with Mg 2+ resulted in high levels of Abl thiophosphorylation, even in the presence of micromolar concentrations of ATP, a development that could allow the study of thiophosphorylation in cell extracts [15].…”

“…This was attributed to the important role of γ-phosphoryl bonding and salt bridging in the Csk reaction transition state [10]. Similarly, we showed that an SH2-binding peptide could be thiophosphorylated by Hck kinase in the presence of cobalt [16]. …”

“…This has facilitated their use in proteomic investigations of kinase activity, where some phosphoproteins are intrinsically unstable, or are modified at low stoichiometry [15]. The thiophosphoryl group can also be further functionalized for proteomic studies [9], or for specific “caging” reactions to produce molecules that are released upon photolysis [16]. …”

“…This is particularly true for tyrosine kinases. To circumvent this problem, we and others have shown that tyrosine kinases can catalyze thiophosphorylation of peptide substrates in the presence of divalent transition metals (e.g., Co 2+ or Ni 2+ ) in the reaction buffer rather than magnesium [10, 16]. This is thought to be due to the increased relative affinity of Co 2+ or Ni 2+ toward sulfur in nucleotide complexes, as compared with Mg 2+ , which has a strong preference for binding oxygen over sulfur [17, 18].…”