Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain

Antonella Scaglione, Laura Monteonofrio, Giacomo Parisi, Cristina Cecchetti, Francesca Siepi, Cinzia Rinaldo, Alessandra Giorgi, Daniela Verzili, Carlotta Zamparelli, Carmelinda Savino, Silvia Soddu, Beatrice Vallone, Linda Celeste Montemiglio

Research output: Contribution to journalArticle

Abstract

The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP-competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross-talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.

Original languageEnglish
Pages (from-to)725-737
Number of pages13
JournalProtein Science
Volume27
Issue number3
DOIs
Publication statusPublished - Mar 1 2018

Fingerprint

Homeodomain Proteins
Phosphorylation
Protein Kinases
Plasticity
Phosphotransferases
Chemical activation
Oligomerization
Denaturation
Lysine
Gel Chromatography
Tyrosine
Conformations
Assays
Catalytic Domain
Substitution reactions
Thermodynamic properties
Hot Temperature
Adenosine Triphosphate
Gels
Cells

Keywords

  • activation-loop phosphorylation
  • homeodomain interacting protein kinase 2 (HIPK2)
  • posttranslational modification
  • protein kinase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Scaglione, A., Monteonofrio, L., Parisi, G., Cecchetti, C., Siepi, F., Rinaldo, C., ... Montemiglio, L. C. (2018). Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain. Protein Science, 27(3), 725-737. https://doi.org/10.1002/pro.3367

Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain. / Scaglione, Antonella; Monteonofrio, Laura; Parisi, Giacomo; Cecchetti, Cristina; Siepi, Francesca; Rinaldo, Cinzia; Giorgi, Alessandra; Verzili, Daniela; Zamparelli, Carlotta; Savino, Carmelinda; Soddu, Silvia; Vallone, Beatrice; Montemiglio, Linda Celeste.

In: Protein Science, Vol. 27, No. 3, 01.03.2018, p. 725-737.

Research output: Contribution to journalArticle

Scaglione, A, Monteonofrio, L, Parisi, G, Cecchetti, C, Siepi, F, Rinaldo, C, Giorgi, A, Verzili, D, Zamparelli, C, Savino, C, Soddu, S, Vallone, B & Montemiglio, LC 2018, 'Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain', Protein Science, vol. 27, no. 3, pp. 725-737. https://doi.org/10.1002/pro.3367
Scaglione A, Monteonofrio L, Parisi G, Cecchetti C, Siepi F, Rinaldo C et al. Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain. Protein Science. 2018 Mar 1;27(3):725-737. https://doi.org/10.1002/pro.3367
Scaglione, Antonella ; Monteonofrio, Laura ; Parisi, Giacomo ; Cecchetti, Cristina ; Siepi, Francesca ; Rinaldo, Cinzia ; Giorgi, Alessandra ; Verzili, Daniela ; Zamparelli, Carlotta ; Savino, Carmelinda ; Soddu, Silvia ; Vallone, Beatrice ; Montemiglio, Linda Celeste. / Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain. In: Protein Science. 2018 ; Vol. 27, No. 3. pp. 725-737.
@article{5278ffe4d43d438fa48ab5f4a7a70b08,
title = "Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain",
abstract = "The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP-competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross-talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.",
keywords = "activation-loop phosphorylation, homeodomain interacting protein kinase 2 (HIPK2), posttranslational modification, protein kinase",
author = "Antonella Scaglione and Laura Monteonofrio and Giacomo Parisi and Cristina Cecchetti and Francesca Siepi and Cinzia Rinaldo and Alessandra Giorgi and Daniela Verzili and Carlotta Zamparelli and Carmelinda Savino and Silvia Soddu and Beatrice Vallone and Montemiglio, {Linda Celeste}",
year = "2018",
month = "3",
day = "1",
doi = "10.1002/pro.3367",
language = "English",
volume = "27",
pages = "725--737",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Cold Spring Harbor Laboratory Press",
number = "3",

}

TY - JOUR

T1 - Effects of Y361-auto-phosphorylation on structural plasticity of the HIPK2 kinase domain

AU - Scaglione, Antonella

AU - Monteonofrio, Laura

AU - Parisi, Giacomo

AU - Cecchetti, Cristina

AU - Siepi, Francesca

AU - Rinaldo, Cinzia

AU - Giorgi, Alessandra

AU - Verzili, Daniela

AU - Zamparelli, Carlotta

AU - Savino, Carmelinda

AU - Soddu, Silvia

AU - Vallone, Beatrice

AU - Montemiglio, Linda Celeste

PY - 2018/3/1

Y1 - 2018/3/1

N2 - The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP-competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross-talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.

AB - The dual-specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis-auto-phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological-like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP-competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross-talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.

KW - activation-loop phosphorylation

KW - homeodomain interacting protein kinase 2 (HIPK2)

KW - posttranslational modification

KW - protein kinase

UR - http://www.scopus.com/inward/record.url?scp=85042218346&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85042218346&partnerID=8YFLogxK

U2 - 10.1002/pro.3367

DO - 10.1002/pro.3367

M3 - Article

AN - SCOPUS:85042218346

VL - 27

SP - 725

EP - 737

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 3

ER -