ABSTRACT: The phosphorylation and dephosphorylation of the NF-AT family of transcription factors play a key role in the activation of T lymphocytes and in the control of the immune response. The mechanistic aspects of NF-AT4 phosphorylation by protein kinase CK1 have been studied in this work with the aid of a series of 27 peptides, reproducing with suitable modifications the regions of NF-AT4 that have been reported to be phosphorylated by this protein kinase. The largest parent peptide, representing the three regions A, Z, and L spanning amino acids 173-218, is readily phosphorylated by CK1 at seryl residues belonging to the A2 segment, none of which fulfill the canonical consensus sequence for CK1. An acidic cluster of amino acids in the linker region between domains A and Z is essential for high-efficiency phosphorylation of the A2 domain, as shown by the increase in K m caused by a deletion of the linker region or a substitution of the acidic residues with glycines. Individual substitutions with alanine of each of the five serines in the A2 domain (S-177, S-180, S-181, S-184, and S-186) reduce the phosphorylation rate, the most detrimental effect being caused by Ser177 substitution which results in a 10-fold drop in V max . On the contrary, the replacement of Ser177 with phosphoserine triggers a hierarchical effect with a dramatic improvement in phosphorylation efficiency, which no longer depends on the linker region for optimal efficiency. These data are consistent with a two-phase phosphorylation mechanism of NF-AT4 by CK1, initiated by the linker region which provides a functional docking site for CK1 and allows the unorthodox phosphorylation of Ser177; once achieved, this phosphoserine residue primes the phosphorylation of other downstream seryl residues, according to a hierarchical mechanism typically exploited by CK1. The large number of protein kinases in eukaryotes, with over 800 genes found in the human genome (1), raises multiple questions as to the function and specificity of these important enzymes. In recent years, several laboratories, including ours, have approached the study of the substrate specificity of protein kinases. These studies have concentrated on the analysis of the amino acid sequences surrounding the immediate vicinity of the sites that are phosphorylated in vivo and in vitro by specific kinases and on the preparation of synthetic peptides that contain these sequences and serve as substrates for these particular enzymes (2-5). These studies have been very useful in determining the consensus sequence recognized preferentially by the active center of these kinases and in predicting the domains of new proteins that are probably phosphorylated by these enzymes. In addition, this approach has allowed us to design several peptides that are highly specific for kinases and that can be employed in assaying for the activity of these kinases in crude extracts of cells and tissues (e.g., refs 5-7). The studies with short peptides, however, demonstrated that these model molecules are sometimes less efficient than the true physiological substrates. In addition, several sequences that contain the defined consensus for phosphorylation by these kinases are not phosphorylated in the native proteins. Conversely, atypical sites that are not acted upon in model peptides serve as good substrates within the context of whole proteins (5). These results clearly indicate that the phosphorylation of proteins by protein kinases involves recognition and interactions that go beyond the immediate vicinity of the acceptor serines or threonines in the substrates. The recent discovery that several protein kinases recognize "docking sites" which are distant from the phosphorylatable residues in their protein substrates constitutes an important step toward the understanding of some of the complexities that provide specificity in kinase-protein substrate interactions (8)
