Characterization of Substrates That Have a Differential Effect on Saccharomyces cerevisiae Protein Kinase A Holoenzyme Activation

Abstract

The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s ؊1 . We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P؉1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P؉1 is not critical. The best substrate has in position P؉4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates.Protein phosphorylation is one of the most fundamental mechanisms for signal transduction. The specificity of signal transduction depends upon the ability of each kinase to precisely phosphorylate particular sites on specific substrate proteins. Protein kinase A (PKA) 3 is one of the best characterized members of this protein family (1). Phosphorylation of its substrate targets is known to be critical for regulating a multitude of cellular processes, including metabolism, gene transcription, ion flux, growth, and cell death (2). The specificity in phosphorylation is determined by at least two major factors: peptide specificity and substrate and kinase localization. The effectiveness of protein phosphorylation by protein kinases in general, including PKA, is believed to depend on the primary structure of the protein around the phosphorylation site; synthetic peptides have thus been used to define the consensus phosphorylation sequences for PKA (3-5). The basic consensus phosphorylation sites for this enzyme are Arg-Arg-X-(Ser/Thr), (Arg/Lys)-X-X-(Ser/Thr), and (Arg/Lys)-X-(Ser/ Thr) (3, 4, 6, 7).The molecular recognition of the peptide sequence surrounding the phosphorylated residue of a substrate usually is referred to as the "peptide specificity" of a protein kinase. The phosphorylated residue is conventionally named P0, the substrate residues immediately N-and C-terminal to the P0 position as PϪ1 and Pϩ1, r...

Regulation of PKA activity by an autophosphorylation mechanism in Saccharomyces cerevisiae

“…Given the charges in the residues involved we hypothesize that allowing backbone flexibility (either in a model or in solution), this distance could be reduced improving the interaction. As a support for this prediction, it has been reported that when the natural arginine residue at P-5 of Nth1-1 was replaced by an acidic residue, the peptide showed lower phosphorylation as determined by peptide array phosphorylation assay [28].…”

“…The kinase activity of purified Tpk1, tpk1 S179A and tpk1 S179D subunits were performed under standard assay conditions except for the Nth1-1 peptide GRQRRLSSLSEF used as substrate [28]. The reaction was started by mixing the samples with assay mixture.…”

“…Cellulose-bound peptide arrays were automatically prepared according to standard SPOT synthesis protocols by using a SPOT synthesizer (Abimed Analysentechnik) by Susan Taylor's laboratory, Department of Chemistry and Biochemistry, University of California at San Diego. The phosphorylation reaction was performed in an incubation mixture containing 0.2 mg/ml BSA, 100 μM ATP, 200 μCi of [γ -32 P]ATP and 150 pmol of Tpk1 subunits for 1 h at 30 • C [28]. The membrane was rinsed, dried and subjected to digital image analysis (Bio-Imaging Analyzer BAS-1800II and Image Gauge 3.12, Fujifilm).…”

“…The effect of the negative charge on Ser 179 on Tpk1 over its kinetic properties was measured by comparing tpk1 S179A activity with tpk1 S179D activity using the peptide substrate NTH1-1 [28]. Figure 6(A) shows that tpk1 S179D has a 25-fold increase in V max , when using the peptide NTH1-1 as a substrate, when compared with tpk1 S179A .…”

Deciphering Enzyme Function Using Peptide Arrays

“…2d) [44] and substitutional libraries (see Fig. 2e) [27,86,[113][114][115][116] enable comprehensive characterisation of substrates on peptide arrays.…”