Recognition of physiological phosphorylation sites by p21-activated kinase 4

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Serine/threonine protein kinases regulate cellular signaling by selectively phosphorylating substrates.
• p21-activated kinase (PAK) family members, particularly PAK4, exhibit a strong preference for serine over threonine phosphorylation.
• LIM domain kinase 1 (LIMK1) is a notable exception, phosphorylated by PAK4 at threonine (Thr508) to enhance its activity.

Purpose of the Study:

• To elucidate the molecular and kinetic basis of PAK4 substrate selectivity.
• To compare PAK4's recognition of the threonine site in LIMK1 with a serine substrate, β-catenin.
• To understand how phosphoacceptor identity influences kinase activity and substrate recognition.

Main Methods:

• X-ray crystallography to determine PAK4-peptide complex structures (LIMK1 and β-catenin peptides).
• Kinase activity assays to measure catalytic efficiency and K_m values.
• Site-directed mutagenesis (T508S mutation in LIMK1).

Main Results:

• PAK4 DFG+1 residue orientation differs between LIMK1 and β-catenin peptides, indicating altered substrate binding.
• Phosphoacceptor identity affects catalytic efficiency but not K_m for both substrates.
• A T508S mutation in LIMK1 significantly enhances its phosphorylation efficiency by PAK4.

Conclusions:

• PAK4 exhibits suboptimal binding to LIMK1's threonine site compared to β-catenin's serine site.
• The differential binding and catalytic efficiency suggest a regulatory mechanism for controlling LIMK1 phosphorylation dynamics by PAK4.
• This suboptimal phosphorylation may serve as a control point for cellular signaling pathways involving PAK4 and LIMK1.