Distal Recognition Sites in Substrates Are Required for Efficient Phosphorylation by the cAMP-Dependent Protein Kinase

Protein kinases are important mediators of signal transduction in eukaryotic cells, and identifying the substrates of these enzymes is essential for a complete understanding of most signaling networks. In this report, novel substrate-binding variants of the cAMP-dependent protein kinase (PKA) were used to identify substrate domains required for efficient phosphorylation in vivo. Most wild-type protein kinases, including PKA, interact only transiently with their substrates. The substrate domains identified were distal to the sites of phosphorylation and were found to interact with a C-terminal region of PKA that was itself removed from the active site. Only a small set of PKA alterations resulted in a stable association with substrates, and the identified residues were clustered together within the hydrophobic core of this enzyme. Interestingly, these residues stretched from the active site of the enzyme to the C-terminal substrate-binding domain identified here. This spatial organization is conserved among the entire eukaryotic protein kinase family, and alteration of these residues in a second, unrelated protein kinase also resulted in a stable association with substrates. In all, this study identified distal sites in PKA substrates that are important for recognition by this enzyme and suggests that the interaction of these domains with PKA might influence specific aspects of substrate binding and/or release.PROTEIN kinases are key mediators of signal transduction in all eukaryotic cells. Each protein kinase modifies a distinct set of substrates, and the biological consequences of activating any kinase are the result of the collective actions of these target proteins (Hunter 2000; Manning et al. 2002). The ability to identify substrates is therefore essential for a complete understanding of most signaling pathways. Unfortunately, this identification process tends to be difficult, and few physiologically relevant targets are known for most protein kinases (Manning and Cantley 2002; Johnson and Hunter 2005). This situation may be changing as a number of innovative approaches to this problem have been developed in recent years (reviewed in Ptacek and Snyder 2006; Deminoff and Herman 2007; Ubersax and Ferrell 2007).This article is focused on the cAMP-dependent protein kinase (PKA) from the budding yeast, Saccharomyces cerevisiae. The PKA enzyme is found in all eukaryotes and is one of the most intensely studied members of this protein family (Taylor et al. 2005). PKA was the first protein kinase structure to be described, and its structure has provided essential insights into the general organization and catalytic mechanism of these enzymes (Knighton et al. 1991; Smith et al. 1999). Subsequent work has illustrated the conserved nature of the protein kinase core and the different ways that the activity of these enzymes can be regulated (Hunter 2000; Huse and Kuriyan 2002; Kannan and Neuwald 2005). In S. cerevisiae, PKA activity is a key regulator of cell growth and the response to environmental stress (Toda et al. 1985; Thevelein and De Winde 1999; Herman 2002; Schneper et al. 2004). We are interested in understanding the role of PKA in these processes and have identified a number of substrates for this enzyme (Howard et al. 2003; Chang et al. 2004; Budovskaya et al. 2005; Deminoff et al. 2006). One of the approaches used for this identification took advantage of PKA variants that exhibit a stable binding to substrate proteins (Deminoff et al. 2006). This binding is novel as most wild-type protein kinases, including PKA, interact only transiently with their substrates (Manning and Cantley 2002). Interestingly, one of these PKA variants was altered at a residue that is conserved in all protein kinases, suggesting that it might be possible to generate substrate-binding versions of other enzymes in this family.These variants of PKA were used here to explore the nature of the protein kinase–substrate interaction. These studies identified substrate domains distal to the sites of phosphorylation that were required for efficient recognition by the wild-type PKA, both in vitro and in vivo. These substrate domains were found to interact with a C-terminal region of PKA that is itself removed from the active site of the enzyme. A systematic mutagenesis of PKA identified additional residues that, when altered, resulted in a stable association with substrates. These latter residues are in close proximity in the three-dimensional structure and may link the active site with this C-terminal substrate-binding domain of PKA. Finally, we show that similar alterations within a second protein kinase, the mammalian double-stranded RNA-dependent protein kinase (PKR), also led to an increased affinity for substrates. In all, the data suggest that the interactions described here may be generally important for protein kinase function and models that explain potential roles for these substrate domains are discussed.