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. 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.
Zhu
Guozhi
G
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Fujii
Koichi
K
Belkina
Natalya
N
Liu
Yin
Y
James
Michael
M
Herrero
Juan
J
Shaw
Stephen
S
D019921:Biotinylation; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry*; D013379:Substrate Specificity
D010455:Peptides; D020033:Protein Isoforms; D011392:Proline; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases