The KLDpT activation loop motif is critical for MARK kinase activity.

Marc Montminy,John R. Yates,Tim Sonntag,James J. Moresco,Hemant K. Paudel

doi:10.1371/journal.pone.0225727

Marc Montminy, John R. Yates + Show 3 more

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https://doi.org/10.1371/journal.pone.0225727

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Abstract

MAP/microtubule-affinity regulating kinases (MARK1-4) are members of the AMPK family of Ser/Thr-specific kinases, which phosphorylate substrates at consensus LXRXXSXXXL motifs. Within microtubule-associated proteins, MARKs also mediate phosphorylation of variant KXGS or ζXKXGSXXNΨ motifs, interfering with the ability of tau and MAP2/4 to bind to microtubules. Here we show that, although MARKs and the closely related salt-inducible kinases (SIKs) phosphorylate substrates with consensus AMPK motifs comparably, MARKs are more potent in recognizing variant ζXKXGSXXNΨ motifs on cellular tau. In studies to identify regions of MARKs that confer catalytic activity towards variant sites, we found that the C-terminal kinase associated-1 (KA1) domain in MARK1-3 mediates binding to microtubule-associated proteins CLASP1/2; but this interaction is dispensable for ζXKXGSXXNΨ phosphorylation. Mutational analysis of MARK2 revealed that the N-terminal kinase domain of MARK2 is sufficient for phosphorylation of both consensus and variant ζXKXGSXXNΨ sites. Within this domain, the KLDpT activation loop motif promotes MARK2 activity both intracellularly and in vitro, but has no effect on SIK2 activity. As KLDpT is conserved in all vertebrates MARKs, we conclude that this sequence is crucial for MARK-dependent regulation of cellular polarity.

Highlights

Introduction5’ AMP-activated protein kinase (AMPK) family members share a high degree of sequence similarity within the N-terminal kinase domains [1], but their regulatory C-termini are far more divergent
We considered that microtubule-affinity regulating kinases (MARKs)-specific regions might modulate zXKXGSXXNC-directed specificity by bringing tau and Microtubule Associated Proteins 2 and 4 (MAP2/4) substrates into physical proximity with the catalytic domain
The S2-M2 hybrid protein was destabilized in comparison to SIK2 and had weaker membrane localization relative to MARK2 (Fig 2E), S2-M2 phosphorylated cyclic AMP (cAMP)-regulated transcriptional coactivators (CRTCs)/ histone deacetylases (HDACs) in a Fsk-resistant manner (Fig 2D). These results indicate that SIK2 and MARK2 are distinguished by their relative sensitivity to cAMP, subcellular localization, and interaction with CLASP1/2 proteins

Summary

Introduction

5’ AMP-activated protein kinase (AMPK) family members share a high degree of sequence similarity within the N-terminal kinase domains [1], but their regulatory C-termini are far more divergent. The most closely related subfamilies of salt-inducible kinases (SIKs; SIK1-3) and MAP/microtubule-affinity regulating kinases (MARKs; MARK1-4) can both phosphorylate cAMP-regulated transcriptional coactivators (CRTCs; CRTC1-3) as well as class IIA histone deacetylases (HDACs; HDAC4,5,7,9) at conserved AMPK substrate motifs, promoting phosphorylation-dependent 14-3-3 protein interactions and cytoplasmic sequestration [9,10,11,12,13] Consistent with their divergent regulatory C-terminal regions, SIK, but not MARK, activity is inhibited by increases in cyclic AMP (cAMP) signaling, where protein kinase A (PKA)-mediated phosphorylation induces 14-3-3 protein binding and inhibition of SIK catalytic activity [11]. These results point to vertebrate MARKs as major regulators of tau and MAPs binding to microtubules

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