Src binds cortactin through an SH2 domain cystine-mediated linkage.

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Tyrosine-kinase signaling is vital for cellular functions.
• Src homology (SH)2 domains bind phosphorylated tyrosines to relay signals.
• Cortactin, a cytoskeletal protein, interacts with SH2-domain proteins to regulate cell motility.

Purpose of the Study:

• To elucidate the unknown mechanism of Src kinase interaction with cortactin.
• To investigate the role of cystine bonding in Src-cortactin interaction.
• To understand the functional implications of this novel binding mode.

Main Methods:

• Demonstrated cystine bonding between Src C185 and cortactin C112/246 using interaction studies and tandem MS/MS sequencing.
• Utilized reducing agents to assess the impact on binding and phosphorylation.
• Performed mutational studies to evaluate the necessity of the cystine binding interface.

Main Results:

• Src binds cortactin via cystine bonding, independent of tyrosine phosphorylation.
• Reducing agents disrupt Src-cortactin binding, cortactin phosphorylation, and SH2 domain interaction.
• Mutations in the cystine binding interface impair Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation.

Conclusions:

• Identified a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin.
• This cystine bond-mediated interaction is crucial for cortactin phosphorylation, cell migration, and invadopodia formation.
• Suggests a widespread alternative signaling mechanism for cysteine-containing SH2 domains beyond tyrosine phosphorylation.