Activation Mechanisms of Conventional Protein Kinase C Isoforms Are Determined by the Ligand Affinity and Conformational Flexibility of Their C1 Domains

Abstract

The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKC␣ and PKC␥, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The C1A and C1B domains of PKC␣ have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. In contrast, the C1A and C1b domains of PKC␥ have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKC␣, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKC␥. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKC␣, neither the C1A nor the C1B domain of PKC␥ is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKC␣ did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKC␥ rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains. Protein kinase C (PKC)1 are a family of serine/threonine kinases that mediate numerous cellular processes (1, 2). 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). Regulatory domains of both conventional and novel PKCs contain tandem C1 (C1A and C1B) domains and a C2 domain. The C1 domain (ϳ50 residues) is a cysteine-rich compact structure that contains five short ␤ strands, a short helix, and two zinc ions (3-7), whereas the C2 domain (ϳ130 residues) is composed of eight-stranded antiparallel ␤ strands (5, 8 -10) and interconnecting loops some of which are involved in Ca 2ϩ -dependent membrane binding. The C1 domain was first identified as the interaction site for diacylglycerol (DAG) and phorbol ester in PKCs (11), but it was subsequently found in other proteins with diverse functions, including protein kinase D (PKD/PKC), chimaerin, Ras-GRP, DAG kinases, and Raf-1 kinase (4, 6, 7). ...

Protein kinase C as a stress sensor

“…Although PKCγ is also a conventional isoform, the conformational flexibility of the C1A and C1B domains in PKCγ were shown to be quite flexible compared to PKCα. In fact, PKCγ translocated to the plasma membrane in the presence of Ca 2+ concentrations which were insufficient to activate PKCα [35].…”

Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

“…PKC is usually cytoplasmic and inactive and translocates to membranes upon binding of lipids and Ca 2ϩ (28,29). But as reported previously (2,23), PKC␥ will translocate with only a DAG signal. PKCs can bind to a scaffold protein, such as 14-3-3, which is thought to bind in the cytoplasm and can either activate or inhibit an individual PKC isoform (26).…”

“…PKC␥ has two C1 domain repeats, C1A and C1B. However, unlike the classical PKC␣ isoform, both the C1A and C1B domain have high affinity for DAG, even at low Ca 2ϩ levels (23). This enzyme can be activated by very low DAG levels (2).…”

Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions

“…PKCγ is a unique isoform of classical PKC which has an exposed C1B domain which binds diacylglycerol (DAG) and is oxidized to form disulfide bonds when the cells are exposed to hydrogen peroxide or other oxidative stress Lin et al, 2003a;Ananthanarayanan et al, 2003). Inactive PKCγ is always associated with 14-3-3 protein in the cytosol by binding to the PKCγ C1B domain where most SCA14 mutations occur (Nguyen et al, 2004).…”

“…The C1 domain is a diacylglycerol (DAG) binding domain, while calcium binds to the C2 domain. Unlike other conventional PKC's, the C1B subdomain of the PKCγ protein is always exposed and calcium is not required for activation of this enzyme (Ananthanarayanan et al, 2003;Lin et al, 2003a). We have demonstrated that the PKCγ C1B subdomain is oxidized when cells are treated with H 2 O 2 which results in formation of disulfide bonds and activation of the enzyme.…”