Contribution of the C1A and C1B Domains to the Membrane Interaction of Protein Kinase C † Jennifer Giorgione, ‡ Michelle Hysell, § Daniel F. Harvey, § and Alexandra C. Newton* ,‡ Department of Pharmacology and Department of Chemistry and Biochemistry, UniVersity of California at San Diego, La Jolla, California 92093-0640 ReceiVed June 12, 2003 ABSTRACT: The hallmark for protein kinase C activation is its “translocation” to membranes following generation of lipid second messengers. This translocation is mediated by the C1 and C2 domains, two membrane-targeting modules, whose engagement on membranes provides the energy for an activating conformational change in which an autoinhibitory pseudosubstrate sequence is released from the active site. Novel and conventional protein kinase C isozymes contain a tandem repeat of C1 domains, the C1A and C1B, which each contain a binding pocket for phorbol esters/diacylglycerol. This study addresses the contribution of the C1A and C1B domains in the regulation of protein kinase C’s membrane interaction using bisfunctional (dimeric) phorbol myristate acetate (PMA) molecules. We show that dimeric bisphorbols are an order of magnitude more effective at recruiting full-length PKC II to membranes compared with monomeric PMA and that the effectiveness of the interaction depends on the nature and length of the cross-link between the PMA moieties. Most effective were dimeric phorbol 12-acetate 13-esters linked at the 13 position with a 14 carbon spacer. The increased potency of dimeric phorbol esters is reduced if either the C1A or C1B domains are mutated so that they are unable to bind PMA, if one moiety of the dimer contains a nonfunctional phorbol, or if the binding to the isolated C1B domain is measured. Thus, the increased potency of the dimeric phorbol esters results primarily from their ability to engage, to a limited extent, both C1 modules on the same molecule. Although dimeric phorbols were more potent than monomeric phorbol esters in recruiting protein kinase C to membranes, the magnitude of the increase was still several orders of magnitude lower than what would be predicted on the basis of the reduction in dimensionality that occurs when the first C1 domain is engaged on the membrane. Thus, engaging both domains can be forced but is highly unfavored. In summary, our data reveal that both C1 domains are oriented for potential membrane interaction but only one C1 domain binds ligand in a physiological context. The reversible translocation from the cytosol to the membrane provides a mechanism to regulate the function of diverse signaling proteins. To effect high sensitivity and specificity in regulating this spatial redistribution, many signaling proteins take advantage of two membrane-targeting modules (1, 2). Each module binds membranes with low affinity, but high-affinity binding is achieved when both domains are engaged on the membrane. Reversibility is achieved by having the membrane affinity of one of the modules depend on stimulus-dependent changes in cofactors (e.g., lipid mediators/Ca 2+ ) or protein structure (e.g., phos- phorylation). Perhaps the best characterized example of this is the family of protein kinase C (PKC) 1 isozymes, in which most members are targeted to the plasma membrane in response to signals that promote phospholipid hydrolysis (3- 5). This translocation is mediated by the engagement of membrane-targeting domains on the membrane providing the energy to release an autoinhibitory pseudosubstrate sequence from the active site of PKC, thus allowing substrate binding and phosphorylation (1). PKC family members comprise a carboxyl-terminal kinase domain and amino-terminal regulatory moiety that contains an autoinhibitory sequence and one or two membrane- targeting modules (4, 5). The membrane-targeting modules are of two types, a C1 and a C2 domain, with a ligand- binding and a non-ligand-binding variant of each (2, 6). The C1 domain binds diacylglycerol and its functional analogues, phorbol esters; the C2 domain binds Ca 2+ . It is the composi- tion of the membrane-targeting modules in this regulatory moiety that defines the three subclasses of isozymes. Conventional isozymes (R, γ, and the alternatively spliced I and II) and novel (δ, ǫ, η/L, θ) PKC isozymes contain a tandem repeat of two ligand-binding C1 domains (C1A and C1B) and a Ca 2+ -binding (conventional isozymes) or non-Ca 2+ -binding (novel isozymes) C2 domain. Atypical † This work was supported by National Institutes of Health Grant GM 43154. J.G. was supported in part by a Canadian Institute of Health Research Fellowship. * Corresponding author. Phone: (858) 534-4527. Fax: (858) 534- 6020. E-mail: anewton@ucsd.edu. ‡ Department of Pharmacology, University of California at San Diego. § Department of Chemistry and Biochemistry, University of Cali- fornia at San Diego. 1 Abbreviations: PKC, protein kinase C; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGTA, [ethylenebis(oxyethyleneni- trilo)]tetraacetic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; PDBu, phorbol 12,13-dibutyrate; EtOAc, ethyl acetate. 11194 Biochemistry 2003, 42, 11194-11202 10.1021/bi0350046 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/05/2003