Dataset Information

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity.

ABSTRACT: Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes.

SUBMITTER: Karuna M P

PROVIDER: S-EPMC7696345 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity.

Karuna M Pradhipa P Witte Leonie L Linnemannstoens Karen K Choezom Dolma D Danieli-Mackay Adi A Honemann-Capito Mona M Gross Julia Christina JC

Biomolecules 20201116 11

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition i ...[more]

PMID: 33207719

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Project description:Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser(19), Ser(33), Ser(35), and Thr(68). This precedes autophosphorylation of Thr(383)/Thr(387) in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366-406). We report four novel phosphorylation sites at Ser(372), Thr(378), Thr(389), and Tyr(390). Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr(389) was a prerequisite to phosphorylation at Thr(387) but not at Thr(383). Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser(379), Thr(389), and Thr(383)/Thr(389). Phosphorylation at Tyr(390) was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr(372), Thr(379), Thr(383), Thr(389), Thr(383)/Thr(387), and Thr(383)/Thr(389). Chk2 hyper-phosphorylated species at Thr(383)/Thr(387)/Thr(389) and Thr(383)/Thr(387)/Thr(389)/Tyr(390) relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.

Dataset Information

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity.

Publications

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity.

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