DEFINITION OF OPTIMAL SUBSTRATE RECOGNITION MOTIFS OF CA2-CALMODULIN-DEPENDENT PROTEIN-KINASE-IV AND PROTEIN-KINASE-II REVEALS SHARED AND DISTINCTIVE FEATURES()

The substrate recognition determinants of Ca2+-calmodulin-dependent pr otein kinase (CaMK) TV and CaMKII alpha were investigated using peptid e substrates modeled on the amino acid sequence encompassing Ser-g of synapsin I, For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Ar g, 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 3 43- fold in V-max/K-m,, for CaMKIV and CaMKII alpha, respectively, For both kinases, the nature of the residues occupying the -4, -1, and 4 positions exerts relatively little influence on phosphorylation kine tics, CaMKIV and CaMKII alpha respond differently to substitutions at the -2 and +1 positions, Substitution of Arg at the -2 position with n on-basic residues (Gin or Ala) leads to a-fold decreases in V-max/K-m, ,JK, for CaMKIV, but 17-28-fold increases for CaMKII alpha., Additiona lly, peptides containing Leu, Asp, or Ala at the +1 position are phosp horylated with similar efficiencies by CaMKIV, whereas the Leu-substit uted peptide is preferred by CaMKII alpha (by a factor of 5.8-9.7-fold ), Thus, CaMKIV and CaMKII alpha preferentially phosphorylate substrat es with the motifs: Hyd-X-Arg-X-X-Ser/Thr*, and Hyd-X-Arg-NB-X-Ser*/T hr-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NE a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiologica l substrates during Ca2+-dependent cellular events.