Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein Kinase D

To investigate the phorbol ester-binding properties of the individual cysteine-rich motifs of protein kinase D (PKD), the 52-mer peptides containing each cysteine-rich motif of PKD (PKD-C1A, PKD-C1B) have been synthesized. The [3H]phorbol-12,13-dibutyrate (PDBu) binding to PKD-C1A was affected drastically by incubation temperature while that to PKD-C1B was not. Scatchard analysis of [3H]PDBu binding to both PKD C1 peptides gave dissociation constants of 2.5 ± 0.4 and 2.7 ± 0.8 nM for PKD-C1A and PKD-C1B, respectively, indicating that the two cysteine-rich motifs of PKD are functionally equivalent like those of PKCγ.