Selective Proteolysis to Study the Global Conformation and Regulatory Mechanisms of c-Src Kinase

Abstract

Protein kinase pathways are traditionally mapped by monitoring downstream phosphorylation. Meanwhile, the noncatalytic functions of protein kinases remain under-appreciated as critical components of kinase signaling. c-Src is a protein kinase known to have noncatalytic signaling function important in healthy and disease cell signaling. Large conformational changes in the regulatory domains regulate c-Src's noncatalytic functions. Herein, we demonstrate that changes in the global conformation of c-Src can be monitored using a selective proteolysis methodology. Further, we use this methodology to investigate changes in the global conformation of several clinical and nonclinical mutations of c-Src. Significantly, we identify a novel activating mutation observed clinically, W121R, that can escape down-regulation mechanisms. Our methodology can be expanded to monitor the global conformation of other tyrosine kinases, including c-Abl, and represents an important tool toward the elucidation of the noncatalytic functions of protein kinases.

Figure 1.. c-Src kinase in the closed (PDB: 2SRC) and the open (PDB: 1Y57) global conformations.

The linker connecting the kinase domain to the SH2 domain (colored grey) is shielded in the closed conformation (colored red) and accessible in the open conformation (colored blue).

Figure 2.. Limited proteolysis using thermolysin can interrogate c-Src global conformation.

c-Src constructs (2 μM), or c-Src (2 μM) pretreated with conformation selective inhibitors (10 μM) were incubated with thermolysin (60 nM) and total c-Src levels were monitored over time. Open conformation c-Src is cut by thermolysin at a rate significantly faster than closed c-Src is cut.

Figure 3.. c-Src mutations and their impact on the global kinase conformation.

All measurements were performed in triplicate and the error reported is the standard deviation.

Figure 4.. Trp-121 and Arg-163 are key residues that stabilize the closed conformation of c-Src.

Trp-121 is located in the SH3 domain and binds within a hydrophobic pocket on kinase domain. Arg-163 is located on the SH3 domain and forms a salt-bridge with Asp-368 that stabilizes the closed conformation. Mutations to these residues destabilize the closed conformation: W121R prevents Trp-121 binding within a hydrophobic pocket of the kinase domain; R163W disrupts the salt bridge between Arg-163 and Asp-368.

Figure 5.. Irreversible conformation-selective analogs inhibitors impact inactivation by Csk.

c-Src kinase constructs with Src^Y530F or Src^Y419F (10 μM) were pretreated with irreversible conformation-selective inhibitors (100 μM) and purified. These constructs, Src-DFGO-Irr and Src-CHO-Irr, were incubated with 3-domain Hck or inactivating kinase Csk (60 nM) and initiated with 100 μM ATP. Phosphorylation at Tyr-419 and Tyr-530 were monitored over time. Reversible conformation-selective dasatinib analogs (PDB: 4YBJ and 4YC8) were overlaid with 3-domain c-Src (PDB: 2SRC and 1Y57).

Figure 6.. W121R mutation resists canonical down-regulation.

Opening mutation were introduced onto the Src^SH2Eng constructs and their catalytic activity and global conformation were assessed. Error reported is the standard deviation of three independent measurements.