Definition of optimal substrate recognition motifs of Ca²⁺- calmodulin-dependent protein kinases IV and II reveals shared and distinctive features

The substrate recognition determinants of Ca²⁺ -calmodulin-dependent protein kinase (CaMK) IV and CaMKIIα were investigated using peptide substrates modeled on the amino acid sequence encompassing Set-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease V(max)/K(m) by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99-and 343- fold in V(max)/K(m) for CaMKIV and CaMKIIα, respectively. For both kinases, the nature of the residues occupying the -4, -1, and +4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIα respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in V(max)/K(m) for CaMKIV, but 17-28-fold increases for CaMKIIα. Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu- substituted peptide is preferred by CaMKIIα (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKHα preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca²⁺ -dependent cellular events.