Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein

Autores
Kronberg, Maria Florencia; Giacometti, Romina; Ruiz Herrera, Jose; Passeron, Susana
Año de publicación
2011
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.
Fil: Kronberg, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina
Fil: Giacometti, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina
Fil: Ruiz Herrera, Jose. Instituto Politecnico Nacional. Unidad Irapuato; México
Fil: Passeron, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina
Materia
AUTOPHOSPHORYLATION
PKA
REGULATORY SUBUNIT
YARROWIA LIPOLYTICA
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/60549
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/60549
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric proteinKronberg, Maria FlorenciaGiacometti, RominaRuiz Herrera, JosePasseron, SusanaAUTOPHOSPHORYLATIONPKAREGULATORY SUBUNITYARROWIA LIPOLYTICAhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.Fil: Kronberg, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Giacometti, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Ruiz Herrera, Jose. Instituto Politecnico Nacional. Unidad Irapuato; MéxicoFil: Passeron, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaElsevier Science Inc2011-05info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/60549Kronberg, Maria Florencia; Giacometti, Romina; Ruiz Herrera, Jose; Passeron, Susana; Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein; Elsevier Science Inc; Archives of Biochemistry and Biophysics; 509; 1; 5-2011; 66-750003-9861CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.abb.2011.03.001info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0003986111000993?via%3Dihubinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-10T13:13:00Zoai:ri.conicet.gov.ar:11336/60549instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-10 13:13:01.247CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
title Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
spellingShingle Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
Kronberg, Maria Florencia
AUTOPHOSPHORYLATION
PKA
REGULATORY SUBUNIT
YARROWIA LIPOLYTICA
title_short Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
title_full Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
title_fullStr Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
title_full_unstemmed Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
title_sort Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein
dc.creator.none.fl_str_mv Kronberg, Maria Florencia
Giacometti, Romina
Ruiz Herrera, Jose
Passeron, Susana
author Kronberg, Maria Florencia
author_facet Kronberg, Maria Florencia
Giacometti, Romina
Ruiz Herrera, Jose
Passeron, Susana
author_role author
author2 Giacometti, Romina
Ruiz Herrera, Jose
Passeron, Susana
author2_role author
author
author
dc.subject.none.fl_str_mv AUTOPHOSPHORYLATION
PKA
REGULATORY SUBUNIT
YARROWIA LIPOLYTICA
topic AUTOPHOSPHORYLATION
PKA
REGULATORY SUBUNIT
YARROWIA LIPOLYTICA
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.
Fil: Kronberg, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina
Fil: Giacometti, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina
Fil: Ruiz Herrera, Jose. Instituto Politecnico Nacional. Unidad Irapuato; México
Fil: Passeron, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina
description cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.
publishDate 2011
dc.date.none.fl_str_mv 2011-05
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/60549
Kronberg, Maria Florencia; Giacometti, Romina; Ruiz Herrera, Jose; Passeron, Susana; Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein; Elsevier Science Inc; Archives of Biochemistry and Biophysics; 509; 1; 5-2011; 66-75
0003-9861
CONICET Digital
CONICET
url http://hdl.handle.net/11336/60549
identifier_str_mv Kronberg, Maria Florencia; Giacometti, Romina; Ruiz Herrera, Jose; Passeron, Susana; Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein; Elsevier Science Inc; Archives of Biochemistry and Biophysics; 509; 1; 5-2011; 66-75
0003-9861
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.abb.2011.03.001
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0003986111000993?via%3Dihub
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science Inc
publisher.none.fl_str_mv Elsevier Science Inc
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.993085

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