Interplay of C1 and C2 Domains of Protein Kinase C-α in Its Membrane Binding and Activation

Abstract

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca 2؉ -dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-␣ and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-␣ is responsible for the initial Ca 2؉ -and phosphatidylserine-dependent electrostatic membrane binding of PKC-␣, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp 58 and Protein kinases C (PKCs)1 are a family of serine/threonine kinases that transduce the myriad of signals activating cellular functions and proliferation (1, 2). More than 10 members of the PKC family have been identified by molecular cloning. All PKCs contain an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Based on structural differences in the regulatory domain, PKCs are generally classified into three groups; conventional PKC (␣, ␤I, ␤II, and ␥ subtypes), novel PKC (␦, ⑀, , and subtypes), and atypical PKC ( and subtypes). The regulatory domain of conventional PKCs is composed of two conserved membrane-targeting modules, C1 and C2 domains, as well as a pseudosubstrate region and variable regions. Conventional PKCs are activated by the Ca 2ϩ -dependent translocation of proteins to the membrane containing phosphatidylserine (PS) and diacylglycerol (DAG). Structural (3) and mutational (4, 5) studies have shown that the C2 domain of conventional PKC is responsible for the Ca 2ϩ -dependent translocation of the protein to membranes. It has also been shown that the C1 domain, which is composed of a tandem repeat of cysteine-rich zinc-finger domains (C1a and C1b), is involved in binding of PKC to DAG and its structural analogs, phorbol esters (6 -8). However, the temporal and spatial sequences of membrane targeting and activation of PKC have not been fully elucidated. Furthermore, the interplay of the C1 and C2 domains in these complex processes is poorly understood. Finally, the roles of individual zinc finger domains in the DAGdependent membrane binding and activation of PKC are not well defined. To address these questions, we functionally expressed the isolated C1 and C2 domains of PKC-␣ and measured their vesicle binding and monolayer penetration. We also mutated C1a and C1b domain residues of the native PKC-␣ and measured the effects of mutations on vesicle binding, enzyme activity, and monolayer penetration. Results indicate that the C2 domain of PKC-␣ is responsible for its initial Ca 2ϩ -and PS-dependent membrane bind...

Mechanism of Diacylglycerol-induced Membrane Targeting and Activation of Protein Kinase Cδ

“…Differential DAG Affinity of C1A and C1B Domains-Our previous studies indicated that the C1A domain plays a critical role in the DAG-induced membrane binding and activation of PKC␣ (38,65), whereas both the C1A and C1B domains of PKC␥ participate in these processes (38). This is ascribed to the fact that the C1A domain of PKC␣ has higher DAG affinity, whereas both the C1A and C1B domains of PKC␥ have comparable DAG affinity and conformational flexibility.…”

The Molecular Basis of Differential Subcellular Localization of C2 Domains of Protein Kinase C-α and Group IVa Cytosolic Phospholipase A2

“…PS Ͼ PG Ͼ Ͼ PC) (15). Our structure-function analysis of PKC-␣ (15) and its isolated C2 domain (36) and the crystal structure of PKC-␣-C2 (12) suggested that the PS selectivity derive from the specific recognition of PS by a C2 domain-bound calcium ion and several residues located in the calcium binding loops (Fig.…”

“…Mutagenesis and Protein Expression-The isolated cPLA 2 -␣-C2 and mutants (26) and the isolated PKC-␣-C2 and mutants (15) were expressed and purified as previously described. Mutants of PKC-␣-C2 were generated by the overlap extension PCR (27) using pVL1392-PKC-␣ plasmid as a template (28).…”

“…Most Ca 2ϩ -dependent membrane binding C2 domains have higher affinity for anionic membranes than for zwitterionic ones. In particular, the C2 domains of PKC-␣ (PKC-␣-C2) and phospholipase C-␦1 show selectivity for phosphatidylserine (PS) (15)(16)(17). In contrast, the C2 domains of group IVa cytosolic phospholipase A 2 (cPLA 2 ) (18) and 5-lipoxygenase (19) strongly favor zwitterionic phosphatidylcholine (PC).…”

Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

“…Pioneer studies with the C2A-domain of synaptotagmin I revealed that the C2 domain acts as a Ca 2+ binding motif [6,48,63]. This function has also been demonstrated in several other C2-domaincontaining proteins such as classical PKCs [5,40,41,49], cPLA2 [12,43] and Nedd4 [45] all of which bind to phospholipids in a Ca 2+ -dependent manner. Furthermore, it has recently been found that Ca 2+ forms a bridge between the C2 membrane-binding domain of PKCα and PS [42].…”