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

Noemi Kedei,Susan H Garfield,Yongmei Pu,Peter M Blumberg

doi:10.1074/jbc.m804796200

Abstract

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 PKCdelta 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 deltaC1a and deltaC1b domains potently bound phorbol esters, but the binding of [(3)H]phorbol 12,13-dibutyrate ([(3)H]PDBu) by the deltaC1a domain depended much more on the presence of phosphatidylserine than did that of the deltaC1b domain. In intact PKCdelta, the deltaC1b domain played the dominant role in [(3)H]PDBu binding, membrane translocation, and down-regulation. A contribution from the deltaC1a domain was nonetheless evident, as shown by retention of [(3)H]PDBu binding at reduced affinity, by increased [(3)H]PDBu affinity upon expression of a second deltaC1a domain substituting for the deltaC1b domain, and by loss of persistent plasma membrane translocation for PKCdelta expressing only the deltaC1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the deltaC1b domain to the normal position of the deltaC1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKCdelta was not the primary determinant of its activity.

Highlights

Erated from phosphatidylinositol 4,5-bisphosphate upon the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D [1]
The DAG-C1 domain-membrane interaction is coupled to a conformational change in protein kinase C (PKC), both causing the release of the pseudosubstrate domain from the catalytic site to activate the enzyme and triggering the translocation to the membrane [2]
In Intact PKC␦, the Deletion of Single C1 Domains Indicates That the ␦C1b Domain Confers Much Higher Potency for Phorbol Ester Binding in Vitro Than Does the ␦C1a Domain—Using the PKC␦-GFP plasmid DNA as template, we first deleted either the ␦C1a or the ␦C1b domain to investigate the effect of constructs were expressed, and they were visualized under identical conditions

Summary

Introduction

Erated from phosphatidylinositol 4,5-bisphosphate upon the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D [1]. In addition to the novel and classic PKCs, six other families of proteins have been identified, some of whose members possess DAG/phorbol ester-responsive C1 domains These are the protein kinase D [5], the chimaerin [6], the munc-13 [7], the RasGRP (guanyl nucleotide exchange factors for Ras and Rap1) [8], the DAG kinase [9], and the recently characterized MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) families [10]. Among the drug candidates in clinical trials that target PKC, a number such as bryostatin 1 and PEP005 are directed at the C1 domains of PKC rather than at its catalytic site Both the classic and novel PKCs contain in their N-terminal regulatory region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester [12]. No such difference in selectivity was observed by Irie et al [20]

Methods

Results

Conclusion