Solution Structure of Atypical Protein Kinase C PB1 Domain and Its Mode of Interaction with ZIP/p62 and MEK5

Abstract

Atypical protein kinase C (aPKC) has been implicated in several signaling pathways such as cell polarity, cell survival, and cell differentiation. In contrast to other PKCs, aPKC is unique in having the PB1 (Phox and Bem 1) domain in the N terminus. The aPKC PB1 domain binds with ZIP/p62, Par6, or MEK5 through a PB1-PB1 domain interaction that controls the localization of aPKC. Here, we determined the three-dimensional structure of the PB1 domain of PKC by NMR and found that the PB1 domain adopts a ubiquitin fold. The OPCA (OPR, PC, and AID) motif inserted into the ubiquitin fold was presented as a ␤␤␣ fold in which the side chains of conserved Asp residues were oriented to the same direction to form an acidic surface. This structural feature suggested that the acidic surface of the PKC PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKC -ZIP/p62 complex by mutational analysis. Interestingly, in the PKC PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motifpresenting surface, suggesting dual roles for the PKC PB1 domain in that it could interact with either the conserved lysine residue or the acidic residues on the OPCA motif of the target PB1 domains.As part of the ongoing study of atypical protein kinase C (aPKC), 1 isoform PKC was first cloned in 1988 (1), followed by the cloning of isoform PKC / (2, 3). Now, aPKC has been suggested as playing a crucial role in signal transduction pathways such as cell polarity, cell differentiation, and cell survival (4, 5). In contrast to conventional PKC, aPKC contains only a single C1 domain but is devoid of a C2 domain in the Nterminal regulatory region. A tandem repeat of the C1 domain is known to be a target of diacylglycerol, and the C2 domain is a binding site for Ca 2ϩ . However, because of the lack of a C2 domain and the incomplete C1 domain in aPKC, neither Ca 2ϩ nor diacylglycerol activates aPKC. Instead, aPKC contains a PB1 domain at the N terminus.In PKC signaling, the regulation of both the catalytic activity and the spatial localization of PKC is essential. PKC is known to translocate to a specific region in response to activation signals and then phosphorylate specific target proteins. Such spatial localization of PKC in cells is partially regulated by either PKC-binding scaffold proteins or anchoring proteins (6). In particular, aPKC binds with PB1 domain-containing proteins such as ZIP/p62 (7-9), Par6 (10 -13), or MEK5 (14) through the PB1-PB1 domain interaction. Thus, the PB1-mediated interaction between aPKC and the scaffolding or anchoring proteins plays a crucial role in exerting the biological functions of aPKC (4).ZIP/p62 homologues were isolated in different biological contexts as ZIP, p62, and EBIAP (7,15,16). Recently, it has been demonstrated that ZIP/p62 is involved in the NF B activation pathway in association with aPKC. ZIP/p62 is a scaffold protein of aPKC for the activation of NF B downstream of extracellular signals such as tumor necrosis fac...

Structural and biochemical insights into the homotypic PB1-PB1 complex between PKCζ and p62

“…When the p62-PB1AM mutant was mixed with equimolar amount of the PB1 domain from PKCζ (PKCζ-PB1, residues 12-101), the vast majority of these two PB1 proteins co-migrated in earlier fractions, indicating the formation of a stable complex with 1 : 1 stoichiometry ( Figure 2C). By contrast, the p62-PB1BM mutant does not interact with PKCζ-PB1 as reported [35]. We also examined the effect of the p62AM mutation on the interaction between two full-length proteins using the coimmunoprecipitation assay, and the result reaffirms that the p62AM mutant, engineered to destroy the acidic OPCA motif, does not abolish the binding of p62 to PKCζ ( Figure 2A).…”

Gαq Acts as an Adaptor Protein in Protein Kinase Cζ (PKCζ)-mediated ERK5 Activation by G Protein-coupled Receptors (GPCR)

“…Besides that effector diversity, our report puts forward a novel scaffold role for G␣ q in ERK5 signaling based on its ability to directly interact with both PKC and MEK5. Our data suggest that activation of GPCR would first promote G␣ q /PKC association followed by direct binding or MEK5 to G␣ q , which would in turn favor PKC/MEK5 interaction through their respective PB1 domains (39,40), leading to ERK5 activation (see model in Fig. 6A).…”

Protein kinase Cι: Human oncogene, prognostic marker and therapeutic target

“…Alignment of the sequences of human PB1 domains reveals a unique cysteine residue in aPKCs, Cys69, located within the conserved OPR, PC and AID (OPCA) motif responsible for binding to par6 and p62 ( Figure 3A). Interestingly, Cys69 is located at the binding interface between PKCι and par6 where it interacts with Arg28, a residue within the basic cluster of par6 involved in PKCι binding (27,79) (Figure 3B). Mutation of Cys69 in PKCι to either isoleucine (C69I) or valine (C69V), the two amino acids most frequently seen at this position in other PB1 domains, has no effect on par6 binding (78).…”

“…aPKCs are thought to be coupled to these signaling molecules via protein-protein interactions involving the PB1 domain of aPKC (25). At least three PB1 domain-containing proteins have been identified that bind to aPKCs via PB1-PB1 domain interactions; p62 (18,79), par6 (21,(80)(81)(82), and Mek5 (79,83). p62 is a scaffold protein that links aPKC to NF-κB downstream of extracellular signals such as tumor necrosis factorα, interleukin-1 and the nerve growth factor (72,84,85).…”