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Abstract:

cAMP-dependent protein kinase mediates many extracellular signals in eukaryotes. The compartmentalization of PKA is an important level of control of the specificity of signal transduction mediated by cAMP. Unlike mammalian PKA for which proof insights in the mechanism that controls its localization through anchoring proteins (AKAPs) has been obtained, in the case of Saccharomyces cerevisiae PKA there was little information available. In this work, we present results that demonstrate the isolation and identification of yeast PKA regulatory subunit (Bcy1) associated proteins using a MS-based proteomic analysis and a bioinformatic approach. The verification of some of these interactions was assessed by immunoprecipitation, pull down and co-localization by subcellular fractionation. The key role of positively charged residues present in the interaction domain of the identified proteins was demonstrated. The defined interaction domain has therefore different molecular characteristics than conventional AKAP domains. Finally we assess initial experiments to visualize the physiological relevance of the interaction of both Ira2 and Hsp60 with Bcy1. Bcy1 interacts with Ira2 tethering PKA to the Ras complex and Hsp60 chaperone localizes PKA to mitochondria and has a role in the kinase stability. Biological significance: Our work has an important impact in the field of signal transduction especially of protein kinase A. Components of the cAMP signaling cascade are localized in the cell via scaffold proteins named AKAPs that contribute to the high level specific regulation of the cAMP-PKA-signaling pathway. In the unicellular eukaryote Saccharomyces cerevisiae PKA has a pleiotropic role in the cell and the compartmentalization therefore is key to achieve the specificity in the response. At present all AKAPs have been described in mammals and it is unknown whether functional homologs of mammalian AKAPs exist in yeast. Therefore, it is unknown which molecular features of the mammalian anchoring proteins are general and which are distinctive. We have identified and characterized interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae, through a proteomic and bioinformatic approach. Bcy1 tethering proteins have a domain in which charged positives residues are key for the interaction with regulatory subunit of PKA and Bcy1 N-terminus is important in the interaction. In mammalian AKAPs a hydrophobic amino acid face of an amphipathic α-helix is essential for the high affinity of the binding interaction. The results obtained in this work seem to indicate that the domains identified in the interacting Bcy1 proteins have a structural nature of the interaction different than those defined for mammalian AKAPs-R interaction. Not only positive charged residues are involved as distinctive molecular determinants but also the hydrophobic face of the helix in which they are included was not relevant in the interaction with Bcy1.Even though generally the use of very well characterized models is essential to answer questions, as would be in this case AKAPs from mammals, the study of other alternative models contributes to the building of more universal concepts. © 2014 Elsevier B.V.

Registro:

Documento: Artículo
Título:Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae
Autor:Galello, F.; Moreno, S.; Rossi, S.
Filiación:Departamento de Química Biológica, IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires 1428, Argentina
Palabras clave:Anchoring proteins; Bcy1; PKA; Sacharomyces cerevisiae; bcy1 protein; chaperonin 60; cyclic AMP dependent protein kinase; Ras protein; Saccharomyces cerevisiae protein; unclassified drug; Bcy1 protein, S cerevisiae; cyclic AMP dependent protein kinase anchoring protein; guanosine triphosphatase activating protein; HSP60 protein, S cerevisiae; IRA2 protein, S cerevisiae; Saccharomyces cerevisiae protein; amino acid; fungal protein; Ira2 protein; protein kinase; article; bioinformatics; cell fractionation; controlled study; enzyme subunit; immunoprecipitation; mass spectrometry; mitochondrion; priority journal; protein analysis; protein domain; protein function; protein isolation; protein localization; protein protein interaction; protein stability; proteomics; regulatory mechanism; Saccharomyces cerevisiae; genetics; metabolism; Article; cell compartmentalization; enzyme stability; eukaryote; mammal; nonhuman; pKa; protein interaction; protein protein interaction; protein subunit; Saccharomyces cerevisiae; signal transduction; yeast; A Kinase Anchor Proteins; Chaperonin 60; GTPase-Activating Proteins; Mitochondria; ras Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomyces cerevisiae
Año:2014
Volumen:109
Página de inicio:261
Página de fin:275
DOI: http://dx.doi.org/10.1016/j.jprot.2014.07.008
Título revista:Journal of Proteomics
Título revista abreviado:J. Proteomics
ISSN:18743919
CAS:cyclic AMP dependent protein kinase; amino acid, 65072-01-7; protein kinase, 9026-43-1; A Kinase Anchor Proteins; Bcy1 protein, S cerevisiae; Chaperonin 60; GTPase-Activating Proteins; HSP60 protein, S cerevisiae; IRA2 protein, S cerevisiae; ras Proteins; Saccharomyces cerevisiae Proteins
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_18743919_v109_n_p261_Galello

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Citas:

---------- APA ----------
Galello, F., Moreno, S. & Rossi, S. (2014) . Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae. Journal of Proteomics, 109, 261-275.
http://dx.doi.org/10.1016/j.jprot.2014.07.008
---------- CHICAGO ----------
Galello, F., Moreno, S., Rossi, S. "Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae" . Journal of Proteomics 109 (2014) : 261-275.
http://dx.doi.org/10.1016/j.jprot.2014.07.008
---------- MLA ----------
Galello, F., Moreno, S., Rossi, S. "Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae" . Journal of Proteomics, vol. 109, 2014, pp. 261-275.
http://dx.doi.org/10.1016/j.jprot.2014.07.008
---------- VANCOUVER ----------
Galello, F., Moreno, S., Rossi, S. Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae. J. Proteomics. 2014;109:261-275.
http://dx.doi.org/10.1016/j.jprot.2014.07.008