A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKC and Par6␣ PB1 domains to a resolution of 1.5 Å. Both PB1 domains adopt a ubiquitin fold. PKC PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6␣ PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKC PB1 and the conserved lysine residue on the Par6␣ PB1. Structural comparison of the PKC and Par6␣ PB1 complex with the p40 phox and p67 phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.

The PB1 (Phox and Bem 1) domain is a recently identified module that mediates formation of a heterodimeric complex with other PB1 domain, e.g. the complexes between the phagocyte oxidase activators p67 phox and p40 phox and between the yeast polarity proteins Bem1p and Cdc24p. These PB1 domains harbor either a conserved lysine residue on one side or an acidic OPCA (OPR/PC/AID) motif around the other side; the lysine of p67 phox or Bem1p likely binds to the OPCA of p40 phox or Cdc24p, respectively, via electrostatic interactions. To further understand molecular recognition by PB1 domains, here we investigate the interactions mediated by proteins presenting both the lysine and OPCA on a single PB1 domain, namely Par6, atypical protein kinase C (aPKC), and ZIP. Par6 and aPKC form a complex via the interaction of the Par6 lysine with aPKC-OPCA but not via that between the aPKC lysine and Par6-OPCA, thereby localizing to the tight junction of epithelial cells. aPKC also uses its OPCA to interact with ZIP, another protein that has a PB1 domain presenting both the lysine and OPCA, whereas aPKC binds via the conserved lysine to MEK5 in the same manner as ZIP interacts with MEK5. In addition, ZIP can form a homotypic complex via the conserved electrostatic interactions. Thus the PB1 domain appears to be a protein module that fully exploits its two mutually interacting elements in molecular recognition to expand its repertoire of protein-protein interactions.

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumorpromoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

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...

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