An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase. Proc Natl Acad Sci USA

Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2005; 102(39):13933-8. DOI: 10.1073/pnas.0501046102
Source: PubMed


Protein kinases are important mediators of much of the signal transduction that occurs in eukaryotic cells. Unfortunately, the identification of protein kinase substrates has proven to be a difficult task, and we generally know few, if any, of the physiologically relevant targets of any particular kinase. Here, we describe a sequence-based approach that simplified this substrate identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. In this method, the evolutionary conservation of all PKA consensus sites in the S. cerevisiae proteome was systematically assessed within a group of related yeasts. The basic premise was that a higher degree of conservation would identify those sites that are functional in vivo. This method identified 44 candidate PKA substrates, 5 of which had been described. A phosphorylation analysis showed that all of the identified candidates were phosphorylated by PKA and that the likelihood of phosphorylation was strongly correlated with the degree of target site conservation. Finally, as proof of principle, the activity of one particular target, Atg1, a key regulator of autophagy, was shown to be controlled by PKA phosphorylation in vivo. These data therefore suggest that this evolutionary proteomics approach identified a number of PKA substrates that had not been uncovered by other methods. Moreover, these data show how this approach could be generally used to identify the physiologically relevant occurrences of any protein motif identified in a eukaryotic proteome.

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Available from: Yelena Budovskaya
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    • "We also examined phosphorylation of PKA sites in YPK3 and KSP1. YPK3 is an AGC kinase that is phosphorylated by PKA in vitro (Budovskaya et al., 2005; Ptacek et al., 2005). KSP1 is a serine/threonine kinase required during filamentous growth in response to nutrient limitation (Bharucha et al., 2008). "
    Dataset: 3475-3

    Full-text · Dataset · May 2015
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    • "Three of the sites are evolutionarily conserved through mono- and dicotyledonous plants and the moss Physcomitrella patens, while two appear to be conserved in nearly all eukaryotes, including budding yeast, fruit flies, nematodes, zebrafish, mouse, and human (Table S2). Given that deep evolutionary conservation of consensus sites is predictive of kinase-substrate relationships [53], we tested whether ADK can be phosphorylated by SnRK1. "
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    ABSTRACT: SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5'-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5'-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "In order to understand if these modifications are in fact not functional or if they are diverging but keeping their function through redundant evolutionary intermediates , we need to determine their functional role (Fig 3). It is possible to use the conservation of PTM sites (Budovskaya et al, 2005; Lam et al, 2010) or the conservation of predicted enzyme-PTM interactions (Tan et al, 2009a) to highlight sites that are more likely functionally important. However, identifying important PTMs through conservation does not predict the function of the modifications and cannot identify species-specific functionally important sites. "
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    ABSTRACT: Protein post-translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post-translational regulation and progress made in the large-scale functional characterization of PTM function.
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