The KLDpT activation loop motif is critical for MARK kinase activity

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• MAP/microtubule-affinity regulating kinases (MARKs) are AMPK family kinases.
• MARKs phosphorylate substrates at consensus and variant motifs, affecting microtubule-associated proteins.
• MARKs interfere with tau and MAP2/4 binding to microtubules.

Purpose of the Study:

• To compare MARKs and salt-inducible kinases (SIKs) in phosphorylating variant motifs.
• To identify MARK regions responsible for variant site phosphorylation.
• To elucidate the role of the KLDpT motif in MARK activity.

Main Methods:

• Comparative substrate phosphorylation assays.
• Analysis of MARK domains, including the KA1 domain.
• Site-directed mutagenesis of MARK2, focusing on the activation loop.
• In vitro and intracellular activity assays.

Main Results:

• MARKs are more potent than SIKs at phosphorylating variant ζXKXGSXXNΨ motifs on tau.
• The C-terminal KA1 domain of MARK1-3 binds CLASP1/2 but is not required for variant phosphorylation.
• The N-terminal kinase domain of MARK2 phosphorylates both consensus and variant sites.
• The KLDpT activation loop motif enhances MARK2 activity but not SIK2 activity.

Conclusions:

• MARKs exhibit specific recognition of variant phosphorylation sites on tau.
• The N-terminal kinase domain and the KLDpT motif are critical for MARK activity.
• The conserved KLDpT motif is essential for MARK-dependent regulation of cellular polarity.