DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain

Jhaisha, Samira Abu; Widowati, Esti; Kii, Isao; Sonamoto, Rie; Knapp, S.; Papadopoulos, Chrisovalantis; Becker, Walter

doi:10.1038/s41598-017-06874-w

Cited by 30 publications

(38 citation statements)

References 43 publications

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“…Wild-type (wt) and mutant GFP-DYRK1A fusion proteins were immunoprecipitated with a GFP antibody from cell extracts of transiently transfected HEK293 cells and subjected to immunoblot analysis. Tyrosine phosphorylation of the activation loop (Y321) was analyzed using an antibody against the activation loop tyrosine in HIPK2 that cross-reacts with the corresponding residue in DYRK1A ( Abu Jhaisha et al, 2017 ). Relative tyrosine phosphorylation was calculated ratio of the pY321 signal relative to total amounts DYRK1A constructs as detected with a DYRK1A specific antibody.…”

Section: Resultsmentioning

confidence: 99%

“…Rabbit polyclonal antibody against phospho-HIPK2 (pTyr361) was purchased from Thermo Fisher Scientific (#PA5-13045; RRID:AB_10987115) and used at 1:1000 dilution for western blot detection. We have previously shown that this antibody is suitable for detection of pY321 in DYRK1A, due to the sequence similarity of the activation loop in HIPK2 and DYRK1A ( Abu Jhaisha et al, 2017 ). Goat polyclonal antibody against GFP was from Rockland Immunochemicals (Limerick, PA, USA; #600-401-215; RRID:AB_828167) and used at 1:1000.…”

Section: Methodsmentioning

confidence: 99%

“…The beads were washed three times with washing buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% Igepal CA-630) and split in two aliquots. One aliquot was used for radiometric assay to determine kinases activities with the peptide substrate DYRKtide as described ( Abu Jhaisha et al, 2017 ). The other aliquot was eluted using Laemmli sample buffer at 95°C for 5 min for immunoblot analysis to assess the relative tyrosine phosphorylation of the DYRK1A constructs.…”

Section: Methodsmentioning

confidence: 99%

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Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

et al. 2018

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Haploinsufficiency of DYRK1A is a cause of a neurodevelopmental syndrome termed mental retardation autosomal dominant 7 (MRD7). Several truncation mutations, microdeletions and missense variants have been identified and result in a recognizable phenotypic profile, including microcephaly, intellectual disability, epileptic seizures, autism spectrum disorder and language delay. DYRK1A is an evolutionary conserved protein kinase which achieves full catalytic activity through tyrosine autophosphorylation. We used a heterologous mammalian expression system to explore the functional characteristics of pathogenic missense variants that affect the catalytic domain of DYRK1A. Four of the substitutions eliminated tyrosine autophosphorylation (L245R, F308V, S311F, S346P), indicating that these variants lacked kinase activity. Tyrosine phosphorylation of DYRK1A-L295F in mammalian cells was comparable to wild type, although the mutant showed lower catalytic activity and reduced thermodynamic stability in cellular thermal shift assays. In addition, we observed that one variant (DYRK1A-T588N) with a mutation outside the catalytic domain did not differ from wild-type DYRK1A in tyrosine autophosphorylation, catalytic activity or subcellular localization. These results suggest that the pathogenic missense variants in the catalytic domain of DYRK1A impair enzymatic function by affecting catalytic residues or by compromising the structural integrity of the kinase domain.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

et al. 2018

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“…We examined phospho-Dyrk1b (p-Y273) levels in C2C12 cells after treatment with cyclopamine. The antibody against p-HIPK2 cross-reacts with the phospho-Y273 residue of the Dyrk1b protein and was previously used to assess Dyrk1b activation (45). WB analysis in cyclopamine treated C2C12 cells revealed higher Dyrk1b auto-phosphorylation upon Shh inhibition compared to untreated cells ( Fig.…”

Section: Dyrk1b Is Activated By Shh Inhibition and Promotes Myotube Fmentioning

confidence: 99%

Dyrk1b is a key Regulatory Kinase Integrating Fgf, Shh and mTORC1 signaling in Skeletal Muscle Development and Homeostasis

Bhat

Narayanan

Fathzadeh

et al. 2020

Preprint

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Dyrk1b: Dual-specificity Tyrosine-regulated kinase 1B SO: sarcopenic obesity MetS: metabolic syndrome myoD1: myoblast determination protein 1 Fgf: Fibroblast growth factor Shh: Sonic Hedgehog mTORC1: mechanistic target of rapamycin C1 4e-bp1: Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 ABSTRACTThe advent of human genetics has provided unprecedented opportunities for discovery of novel disease pathways. Mutations in DYRK1B have been associated with metabolic syndrome and sarcopenic obesity in humans, underscoring the critical role of the encoded protein in skeletal muscle development and homeostasis. By the novel creation of Dyrk1b knockout zebrafish models we demonstrate that Dyrk1b kinase activity is critical for specification of the paraxial myoD. Mechanistically, Dyrk1b mediates and amplifies Fgf signaling in the paraxial domain by the transcriptional suppression of its negative feedback inhibitor sprouty1. In the adaxial myoD domain, Dyrk1b amplifies Shh signaling and partially rescues defects caused by its disruption. The investigations of C2C12 terminal differentiation revealed that Dyrk1b also plays a critical role in myofiber fusion. Combined biochemical and proteomic analysis of C2C12 myoblasts undergoing differentiation showed that Dyrk1b kinase activation is induced by shh inhibition, and triggers differentiation by inhibiting mTOR, subsequent upregulation of 4e-bp1 and induction of autophagy. In conclusion, we demonstrate that Dyrk1b plays a critical role in sustaining myocyte specification and differentiation by integrating Fgf, Shh and mTORC1 signaling pathways. determination, inherited metabolic syndrome. E lateral views, F-G dorsal views, H and I are cross-sections at regions indicated by red lines on G. Red asterisks on F and G indicate the unstained axial domain. Red asterisks on I denote somites, red arrow denotes notochord, NT=neural tube. J,K are group shots of zebrafish embryos stained with anti-sense and the control sense probe. L. Schematic diagram of the dyrk1b locus in the zebrafish genome. Boxes indicate the exons (not drawn to scale). The dotted lines from second exon indicate the translation start site (in red). Guide RNA is highlighted in yellow and PAM region in green. wildtype and dyrk1b+/-sequence are shown, 7bp insertion induced by CRISPR/Cas9 is highlighted in blue. M Schematic showing Dyrk1b CRISPR/Cas9 injection and transmission of induced mutation. N T7 assay with one band for Wildtype and two bands for dyrk1b+/-, indicating the heteroduplex formed by CRISPR/Cas9 activity. O. Sanger sequencing images of amplified segments of wildtype and dyrk1b+/-, demonstrating the site of 7bp insertion (red arrow) and the ensuing frame shift. P. dyrk1b mRNA expression levels (mean + s.e.m) in the wildtype and Dyrk1b+/-zebrafish embryo at 24hpf (*** p≤ 0.001, Students ttest, 2 tailed, n=30 pooled embryos).

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“…This holds particularly true for DYRK1B carrying mutations which were identified in families suffering from an autosomal-dominant form of metabolic syndrome [ 35 ], a disease with prominent adipocyte involvement. The mutations found result in misfolding of the DYRK1B protein and in intracellular aggregation [ 124 ]. It remains to be clarified how these mutations affect the functional integration of DYRK1B into other signaling pathways, but it is intriguing to note that mutant DYRK1B expression reduced GLI2 levels in cultured adipocytes [ 35 ].…”

Section: Class I Dyrks: Dyrk1a and Dyrk1bmentioning

confidence: 99%

Emerging Roles of DYRK Kinases in Embryogenesis and Hedgehog Pathway Control

Singh

Lauth

2017

JDB

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Hedgehog (Hh)/GLI signaling is an important instructive cue in various processes during embryonic development, such as tissue patterning, stem cell maintenance, and cell differentiation. It also plays crucial roles in the development of many pediatric and adult malignancies. Understanding the molecular mechanisms of pathway regulation is therefore of high interest. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) comprise a group of protein kinases which are emerging modulators of signal transduction, cell proliferation, survival, and cell differentiation. Work from the last years has identified a close regulatory connection between DYRKs and the Hh signaling system. In this manuscript, we outline the mechanistic influence of DYRK kinases on Hh signaling with a focus on the mammalian situation. We furthermore aim to bring together what is known about the functional consequences of a DYRK-Hh cross-talk and how this might affect cellular processes in development, physiology, and pathology.

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DYRK1B mutations associated with metabolic syndrome impair the chaperone-dependent maturation of the kinase domain

Cited by 30 publications

References 43 publications

Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

Functional characterization of DYRK1A missense variants associated with a syndromic form of intellectual deficiency and autism

Dyrk1b is a key Regulatory Kinase Integrating Fgf, Shh and mTORC1 signaling in Skeletal Muscle Development and Homeostasis

Emerging Roles of DYRK Kinases in Embryogenesis and Hedgehog Pathway Control

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