Exceptional Disfavor for Proline at the P+1 Position among AGC and CAMK Kinases Establishes Reciprocal Specificity between Them and the Proline-directed Kinases

Abstract

To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulindependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P؉1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P؉1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P؉1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.Phosphorylation is a prevalent modification in cells that controls many functions such as signaling transduction, proliferation and apoptosis. It is estimated that at least one-third of all proteins in eukaryotic cell are phosphorylated at any given time (1, 2). More than 500 human protein kinases have been identified so far (3). A high degree of selectivity in substrate phosphorylation is necessary to maintain functional integrity of this very complicated signaling environment. Precision in phosphorylation is particularly critical when a substrate protein is phosphorylated at two (or more) phosphorylation sites, and those sites 1) are phosphorylated by distinct upstream kinases and 2) confer distinct properties on that substrate. To assure fidelity of signaling in this common situation, each upstream kinase must show high specificity by phosphorylating only the relevant phosphorylation site and not the inappropriate site(s). This requirement poses a major challenge in evolutionary design of kinase peptide specificity, since those upstream kinases are usually recruited to the substrate, and such recruitment can overcome much of the barrier provided by peptide specificity (4).These considerations raise the important issue as to what elements in kinase peptide specificity confer the strongest reciprocal specificity between kinases, i.e. which prevent one kinase from phosphorylating substrates phosphorylated by another. For Ser/Thr kinases, we propose that much of this reciprocal specificity is provided by the evolution of three broad classes (5): basophilic kinases that phosphorylate sites with clustered positive charges, acidophilic kinases that phosphorylate sites with clustered negative charges, and proline-directed kinases that ph...

Cell-cycle–dependent oscillation of GATA2 expression in hematopoietic cells

“…Because S 0 P ϩ1 and T 0 P ϩ1 motifs are likely targets of Cdks or MAP kinases, 35 but not of the other kinases, 53 these results suggested Cdk/cyclin-dependent phosphorylation of Flag⌬NTGATA2 at S 0 P ϩ1 or T 0 P ϩ1 motifs.…”

Protein Kinase CβII-Mediated Phosphorylation of Endothelial Nitric Oxide Synthase Threonine 495 Mediates the Endothelial Dysfunction Induced by FK506 (Tacrolimus)

“…Another inhibitory phosphorylation site on eNOS that may be phosphorylated by a PKC isoform is Ser116. Kou et al (2002) showed that the PKC inhibitor calphostin could reduce eNOS Ser116 phosphorylation; however, others have shown that PKC cannot phosphorylate eNOS Ser116 because it is followed by a proline (Fujii et al, 2004;Zhu et al, 2005). Therefore, although FK506 may be having an indirect effect on Ser116, it is not related directly to the PKC-mediated phosphorylation of eNOS Thr495 induced by FK506 (Fig.…”

Structure and Substrate Specificity of the Pim-1 Kinase

“…This conformation appears to be stabilized in part by an interaction between the backbone carbonyl of the residue immediately upstream (Asp-202) and the side chain of Ser-189 at the start of the activation loop. As noted by Shaw and co-workers (54,55) for other kinases belonging to the AGC and CAMK groups, Pim kinases appear to strongly deselect Pro in the ϩ1 position in substrates. This "proline disfavor" phenomenon is generally attributable to the inability of Pro to hydrogen bond to the carbonyl group of the residue equivalent to Gly-203 in the kinase.…”