A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery

Nevenzal, Hadas; Noach-Hirsh, Meirav; Skornik-Bustan, Or; Brio, Lev; Barbiro-Michaely, Efrat; Glick, Yaïr; Avrahami, Dorit; Lahmi, Roxane; Tzur, Amit; Gerber, Doron

doi:10.1038/s42003-019-0286-9

Cited by 9 publications

(7 citation statements)

References 43 publications

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“…Also, several studies suggested that both ROR1 and ROR2 might be pseudokinases (Gentile et al, 2011;Debebe and Rathmell, 2015). However, recent studies by Nevenzal et al (2019) and Sheetz et al (2020) had found the autophosphorylation activity and autoinhibitory interactions of the TKD in the ROR2 protein from the perspective of highthroughput phosphorylation detection and protein structure. Consistent with results from experiments in vitro, five of our patients carrying the c.1675G > A heterozygous mutation mainly displayed short stature.…”

Section: Discussionmentioning

confidence: 99%

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature

Gui

et al. 2021

Front. Cell Dev. Biol.

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PurposeROR2, a member of the ROR family, is essential for skeletal development as a receptor of Wnt5a. The present study aims to investigate the mutational spectrum of ROR2 in children with short stature and to identify the underlying molecular mechanisms.MethodsWe retrospectively analyzed clinical phenotype and whole-exome sequencing (WES) data of 426 patients with short stature through mutation screening of ROR2. We subsequently examined the changes in protein expression and subcellular location in ROR2 caused by the mutations. The mRNA expression of downstream signaling molecules of the Wnt5a–ROR2 pathway was also examined.ResultsWe identified 12 mutations in ROR2 in 21 patients, including 10 missense, one nonsense, and one frameshift. Among all missense variants, four recurrent missense variants [c.1675G > A(p.Gly559Ser), c.2212C > T(p.Arg738Cys), c.1930G > A(p.Asp644Asn), c.2117G > A(p.Arg706Gln)] were analyzed by experiments in vitro. The c.1675G > A mutation significantly altered the expression and the cellular localization of the ROR2 protein. The c.1675G > A mutation also caused a significantly decreased expression of c-Jun. In contrast, other missense variants did not confer any disruptive effect on the biological functions of ROR2.ConclusionWe expanded the mutational spectrum of ROR2 in patients with short stature. Functional experiments potentially revealed a novel molecular mechanism that the c.1675G > A mutation in ROR2 might affect the expression of downstream Wnt5a–ROR2 pathway gene by disturbing the subcellular localization and expression of the protein.

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Section: Discussionmentioning

confidence: 99%

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature

Gui

et al. 2021

Front. Cell Dev. Biol.

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“…Filamin A, an actin-crosslinking protein, binds to the intracellular PRD of Ror2 and is involved in regulating Ror2-mediated polarized cell migration 9,10 (Figure 1). Although mutations in lysine residues within the putative ATP binding motif in the tyrosine kinase domains of the Ror-family RTKs have been shown to result in some impairment of their functions, [11][12][13] several lines of evidence have suggested that both Ror1 and Ror2 might be pseudokinases. 14,15 Therefore, it remains controversial whether or not Ror1 and Ror2 possess intrinsic kinase activities.…”

Section: Structure and Function Of The Ror-family Rtksmentioning

confidence: 99%

Role of noncanonical Wnt ligands and Ror‐family receptor tyrosine kinases in the development, regeneration, and diseases of the musculoskeletal system

Kamizaki

Endo

Minami

et al. 2020

Developmental Dynamics

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The Ror-family receptor tyrosine kinases (RTKs), consisting of Ror1 and Ror2, play crucial roles in morphogenesis and formation of various tissues/organs, including the bones and skeletal muscles, the so-called musculoskeletal system, during embryonic development, by acting as receptors or coreceptors for a noncanonical Wnt protein Wnt5a. Furthermore, several lines of evidence have indicated that Ror1 and/or Ror2 play critical roles in the regeneration and maintenance of the musculoskeletal system in adults. Considering the

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“…Such in vitro systems enable better control of various biochemical parameters and processes than in vivo systems [ 25 , 26 , 27 , 28 ]. They are regularly used to study gene circuits or reaction cascades [ 29 , 30 , 31 ]. In vitro, proteins, in the form of wild-type proteins or with modifications, can easily be produced recombinantly by the simple addition of the corresponding coding DNA [ 32 , 33 , 34 ].…”

Section: Introductionmentioning

confidence: 99%

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

Khangholi

Finkler

Seemann

et al. 2021

IJMS

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Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

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A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery

Cited by 9 publications

References 43 publications

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature

Heterozygous Recurrent Mutations Inducing Dysfunction of ROR2 Gene in Patients With Short Stature

Role of noncanonical Wnt ligands and Ror‐family receptor tyrosine kinases in the development, regeneration, and diseases of the musculoskeletal system

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

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