Characterization of the Differential Roles of the Twin C1a and C1b Domains of Protein Kinase Cδ

Classic and novel protein kinase C (PKC) isozymes contain two zinc finger motifs, designated C1a and C1b domains, which constitute the recognition modules for the second messenger diacylglycerol (DAG) or the phorbol esters. However, the individual contributions of these tandem C1 domains to PKC function and, reciprocally, the influence of protein context on their function remain uncertain. In the present study, we prepared PKC delta constructs in which the individual C1a and C1b domains were deleted, swapped, or substituted for one another to explore these issues. As isolated fragments, both the delta C1a and delta C1b domains potently bound phorbol esters, but the binding of [H-3] phorbol 12,13-dibutyrate ([H-3]PDBu) by the delta C1a domain depended much more on the presence of phosphatidylserine than did that of the delta C1b domain. In intact PKC delta, the delta C1b domain played the dominant role in [H-3]PDBu binding, membrane translocation, and down-regulation. A contribution from the delta C1a domain was nonetheless evident, as shown by retention of [H-3]PDBu binding at reduced affinity, by increased [H-3]PDBu affinity upon expression of a second delta C1a domain substituting for the delta C1b domain, and by loss of persistent plasma membrane translocation for PKC delta expressing only the delta C1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the delta C1b domain to the normal position of the delta C1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKC delta was not the primary determinant of its activity.