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 calmodulin-dependent 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.
Highlights
Exceptional Disfavor for Proline at the P؉1 Position among AGC and CAMK Kinases Establishes Reciprocal Specificity between Them and the Proline-directed Kinases*
To test these structural hypotheses we have mutationally swapped the toggle residue and toggleregulating residues between Cdk2 and PKC-␦; both constructs are catalytically inactive, indicating that these residues function normally only in the context of other kinase family-related residues. These patterns of conservation of the toggle residue and toggle-regulating residue are fully consistent with their critical role in establishing a major difference between CMGC kinase and AGC/ CAMK kinases. These results demonstrate that Pro at the Pϩ1 position is a veto residue which virtually precludes phosphorylation by multiple AGC and CAMK kinases
Pro at Pϩ1 provides tight control of reciprocal substrate specificity between prolinedirected kinases and AGC/CAMK kinases. These findings for peptides are relevant to proteins, since we find that Pro occurs at a much reduced frequency at the Pϩ1 position among reported substrates for PKA and PKC
Summary
Exceptional Disfavor for Proline at the P؉1 Position among AGC and CAMK Kinases Establishes Reciprocal Specificity between Them and the Proline-directed Kinases*. 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. 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). Proline-directed kinases will eschew substrates of the other two which lack proline at Pϩ1
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
