Interacting proteins of protein kinase A regulatory subunit in Saccharomyces cerevisiae.

UNLABELLED:cAMP-dependent protein kinase mediates many extracellular signals in eukaryotes. The compartmentalization of is an important level of control of the specificity of signal transduction mediated by cAMP. Unlike mammalian duanyu1529 for which proof insights in the mechanism that controls its localization through anchoring proteins (AKAPs) has been obtained, in the case of Saccharomyces cerevisiae duanyu1529 there was little information available. In this work, we present results that demonstrate the isolation and identification of yeast duanyu1529 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 duanyu1529 to the Ras complex and Hsp60 chaperone localizes duanyu1529 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 pathway. In the unicellular eukaryote Saccharomyces cerevisiae duanyu1529 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 duanyu1529 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.

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