The cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) share a strong primary sequence homology within their respective active site regions. Not surprisingly, these enzymes also exhibit overlapping substrate specificities, a feature that often interferes with efforts to elucidate their distinct biological roles. In this report, we demonstrate that PKA and PKG exhibit dramatically different behavior with respect to the phosphorylation of ␣-substituted alcohols. Although PKA will phosphorylate only residues that contain an ␣-center configuration analogous to that found in L-serine, PKG utilizes residues that correspond to both L-and D-serine as substrates. The PKG/PKA selectivity of these substrates is the highest ever reported.Protein kinase cascades are the predominant component of signal transduction pathways (1). These pathways transmit extracellular signals from the plasma membrane to distant intracellular sites in the cytoplasm and nucleus. Estimates have placed the potential number of protein kinases encoded by the mammalian genome at greater than 1,000 (2). Consequently, defining the role of a particular protein kinase can be a daunting task given the size of this enzyme family and the plethora of pathways in which these phosphoryl transfer catalysts serve as participants. This task is rendered even more formidable by the general nature of the reaction catalyzed by these enzymes and by their nearly universal utilization of a common substrate, ATP. However, individual protein kinases presumably do exhibit unique properties in vivo, as exemplified by their ability to catalyze the phosphorylation of specific proteins. This precision is potentially regulated by several different parameters, including (i) the microenvironment to which the enzyme is confined within the cell (3), (ii) the secondary and/or tertiary structure that encompasses the site of phosphorylation on the target protein substrate, (iii) the primary sequence that envelops the phosphorylatable amino acid residue, and (iv) the active site substrate specificity of the protein kinase, which is generally limited to serine, threonine, and/or tyrosine residues (although exceptions are known).Can these four parameters be manipulated to design inhibitors that target specific protein kinases? Peptide-based substrates have been described for a number of protein kinases utilizing data from the specificity parameter (iii) described above (4). These substrate specificity studies have lead to the creation of nonphosphorylatable reversible inhibitors: peptides in which the alcohol-containing amino acid has been replaced by a residue that lacks a hydroxyl moiety (e.g. alanine for serine exchange). Unfortunately, the ultimate utility of this approach is limited by the fact that many protein kinases exhibit overlapping substrate specificities. A case in point is the cAMP-dependent protein kinase (5, 6) and its closely related counterpart, the cGMP-dependent protein kinase (6, 7). Both enzymes exhibit a special affinity for amino acid sequences that con...
The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].
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