Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

Abstract

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this auto-inhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer’s, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

Intramolecular autoinhibition of checkpoint kinase 1 is mediated by conserved basic motifs of the C-terminal kinase–associated 1 domain

“…Kinetic and biophysical studies reveal a high-affinity intramolecular autoinhibitory interaction of the Chk1 kinase domain emanating from the KA1 domain. Extensive site-directed mutagenesis implicates CM1 and CM2 as playing a central role in autoinhibitory interactions, especially basic residues within these regions, pinpointing the likely interface of autoinhibition among all Chk1 orthologs and linking Chk1 autoinhibition to that previously described for MARK1 (16). Intimate knowledge of the mechanism of KA1-mediated Chk1 autoinhibition may lead to novel strategies to modulate activity of this validated oncology target.…”

“…Given that crystal structures of the Chk1 kinase domain show an open "active" conformation (14) (Fig. 1A, bottom gray), and activation loop phosphorylation has not been described for Chk1, we wondered whether the Chk1 KA1 domain could autoregulate the kinase domain (15)(16)(17). C-terminal truncations of human Chk1 result in substantially more active kinase in vitro (14), and kinase and C-terminal domains from the Chk1 ortholog in Xenopus laevis can associate with one another (18), suggesting that the human Chk1 KA1 domain may act as an autoinhibitory domain as recently described for MARK1 (16).…”

“…1A, bottom gray), and activation loop phosphorylation has not been described for Chk1, we wondered whether the Chk1 KA1 domain could autoregulate the kinase domain (15)(16)(17). C-terminal truncations of human Chk1 result in substantially more active kinase in vitro (14), and kinase and C-terminal domains from the Chk1 ortholog in Xenopus laevis can associate with one another (18), suggesting that the human Chk1 KA1 domain may act as an autoinhibitory domain as recently described for MARK1 (16). Recent in vivo FRET experiments indicate that the N and C termini of Chk1 separate following DNA damage or in response to particular KA1 domain mutants (19), and Chk1 is constitutively activated when predicted secondary structure elements of the KA1 domain are disrupted (20).…”

Autoinhibition can identify rare driver mutations and advise pharmacology

“…129 There, the KA1 membrane-binding domain at the C-terminal binds both the N-and C-lobes of the kinase domain with the KA1 domain preventing peptide substrate binding. 129 There, the KA1 membrane-binding domain at the C-terminal binds both the N-and C-lobes of the kinase domain with the KA1 domain preventing peptide substrate binding.…”

“…The autoinhibition mechanism mediated by the KA1 domain has been exquisitely worked out in MARK1. 129 There, the KA1 membrane-binding domain at the C-terminal binds both the N-and C-lobes of the kinase domain with the KA1 domain preventing peptide substrate binding. KA1 residues involved in autoinhibition are those involved in anionic phospholipid binding.…”