Skip to Content
Merck
  • Proline Hydroxylation Primes Protein Kinases for Autophosphorylation and Activation.

Proline Hydroxylation Primes Protein Kinases for Autophosphorylation and Activation.

Molecular cell (2020-07-09)
Sang Bae Lee, Aram Ko, Young Taek Oh, Peiguo Shi, Fulvio D'Angelo, Brulinda Frangaj, Antonius Koller, Emily I Chen, Timothy Cardozo, Antonio Iavarone, Anna Lasorella
ABSTRACT

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Sigma-Aldrich
α-Ketoglutaric acid, 99.0-101.0% (T)
Sigma-Aldrich
L-Ascorbic acid, reagent grade, crystalline
Sigma-Aldrich
3X FLAG® Peptide, lyophilized powder
Millipore
Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
Hexadimethrine bromide, ≥94% (titration)
Sigma-Aldrich
Cobalt(II) chloride hexahydrate, BioReagent, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
Catalase from bovine liver, powder, suitable for cell culture, 2,000-5,000 units/mg protein
Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, ascites fluid, clone B-5-1-2
Roche
Anti-HA-Biotin, High Affinity (3F10), from rat IgG1
Sigma-Aldrich
Doxycycline hydrochloride
Sigma-Aldrich
MG-132, A cell-permeable, potent, reversible proteasome inhibitor (Ki = 4 nM).
Roche
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Protease Inhibitor Cocktail Tablets provided in a glass vial, Tablets provided in a glass vial