An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Autophosphorylation is crucial for protein kinase regulation but its molecular basis is not fully understood.
• c-Src kinase undergoes autophosphorylation, influencing its functional states and interactions.

Purpose of the Study:

• To elucidate the structural and molecular mechanisms governing c-Src autophosphorylation.
• To investigate the roles of specific tyrosine residues (Tyr 419 and Tyr 530) in c-Src regulation.
• To understand how c-Src autophosphorylation impacts its catalytic and non-catalytic functions.

Main Methods:

• Time-resolved kinetics studies to analyze phosphorylation rates.
• X-ray crystallography to visualize intermediate states of autophosphorylation.
• Analysis of cancer-associated c-Src variants with altered C-terminal sequences.

Main Results:

• C-terminal Tyr 530 is a slow autophosphorylation site with intermolecular kinetics.
• Activation-loop Tyr 419 phosphorylation is faster and controls Tyr 530 phosphorylation via a cis-to-trans switch.
• A palindromic phospho-motif near Tyr 530 engages the active kinase, facilitating intermolecular autophosphorylation.
• C-terminal deletions disrupt this motif, impairing Tyr 530 autophosphorylation and causing c-Src dysfunction.

Conclusions:

• A crosstalk exists between c-Src activation loop and C-terminus, regulating kinase activity and substrate function.
• The identified phospho-motif and its interaction with the active kinase are critical for c-Src autophosphorylation and function.
• Dysfunction in this regulatory mechanism, as seen in cancer variants, highlights its importance in normal physiology and disease.