Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions

Abstract

Modular domains mediating specific protein-protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67(phox), which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40(phox) (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40(phox), which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.

Fig. 1. Pinpointing the Cdc24p-binding region on Bem1p. Various portions of Bem1p were tested for binding to Cdc24p by two-hybrid assays (A) and in vitro pull-down assays (B). PB2 represents a novel domain conserved among p47^phox, p40^phox, Bem1p and various other signaling proteins (Sumimoto et al., 1997; Ago et al., 1999). It is also called the PX domain (Ponting, 1996).

Fig. 2. Role for the PC motif in the association of Cdc24p with Bem1p. (A) Pinpointing the Bem1p-binding region on Cdc24p. The C-terminal 75 aa region, which was previously shown to bind to Bem1p (Peterson et al., 1994), was further deleted from either the N- or the C-terminus and examined for two-hybrid interaction with Bem1p. (B) The PC motifs from various proteins are aligned with a tentative consensus sequence shown on the top, in which hydrophobic residues are represented by #. ZIP is also known as p62, p60 or ORCA. (C) Effects of PC motif mutations examined by the yeast two-hybrid assay. A DNA-binding domain fusion plasmid pGBK-Bem1p-(472–551) was co-transformed with an activation domain fusion plasmid pGADg-Cdc24p-(780–854) (WT) or pGADg-Cdc24p-(780–854, D824K/D831R) (mt) into PJ69-2A (Clontech) and SFY526 (Clontech). Following selection on SC-Trp-Leu plates, PJ69-2A transformants were tested for Ade-, His-independent growth (top) and SFY526 cells were examined for β-galactosidase activity by the filter assay using X-gal as the substrate (bottom). (D) Effects of PC motif mutations examined by in vitro pull-down assays. The purified GST–Bem1p-(464–551) fusion protein was incubated with purified MBP–Cdc24p-(780–854) (WT) or MBP–Cdc24p-(780–854, D824K/D831R) (mt). Subsequently, proteins were precipitated with glutathione–Sepharose (left) or amylose resin (right), and were resolved on SDS–PAGE followed by Coomassie Brilliant Blue staining.

Fig. 3. Pinpointing the p67^phox domain that interacts with the PCCR of p40^phox. The inter-SH3 region of p67^phox, which had been shown to bind to the PCCR of p40^phox (Nakamura et al., 1998), was variously deleted from either the N- or the C-terminus and tested for binding to p40^phox by two-hybrid assays (A) or in vitro pull-down assays (B).

Fig. 4. PB1 domain as the binding partners for PCCRs. (A) Alignment of PB1 domains. Pinpointed regions in Figures 1 and 3 are aligned with the corresponding regions of fission yeast scd2 and mouse p67^phox. The lower portion shows additional PB1 domains revealed by PSI-BLAST search. They include members of the atypical protein kinase C family from various species and anonymous hypothetical proteins in the Arabidopsis genome (indicated by their DDBJ/EMBL/Genbank accession Nos). A tentative consensus sequence is shown on the top, where # stands for hydrophobic residues. (B) Effect of PB1 domain mutation in Bem1p. The conserved K482 residue of Bem1p was replaced with alanine, and tested for binding to Cdc24p using two-hybrid (upper) and in vitro pull-down assays (lower). (WT, wild type; mt, mutant). (C) Effect of PB1 domain mutation in p67^phox. The conserved K355 residue was replaced with alanine, and tested for binding to p40^phox using two-hybrid (upper) and in vitro pull-down assays (lower). (WT, wild type; mt, mutant).

Fig. 4. PB1 domain as the binding partners for PCCRs. (A) Alignment of PB1 domains. Pinpointed regions in Figures 1 and 3 are aligned with the corresponding regions of fission yeast scd2 and mouse p67^phox. The lower portion shows additional PB1 domains revealed by PSI-BLAST search. They include members of the atypical protein kinase C family from various species and anonymous hypothetical proteins in the Arabidopsis genome (indicated by their DDBJ/EMBL/Genbank accession Nos). A tentative consensus sequence is shown on the top, where # stands for hydrophobic residues. (B) Effect of PB1 domain mutation in Bem1p. The conserved K482 residue of Bem1p was replaced with alanine, and tested for binding to Cdc24p using two-hybrid (upper) and in vitro pull-down assays (lower). (WT, wild type; mt, mutant). (C) Effect of PB1 domain mutation in p67^phox. The conserved K355 residue was replaced with alanine, and tested for binding to p40^phox using two-hybrid (upper) and in vitro pull-down assays (lower). (WT, wild type; mt, mutant).

Fig. 5. Specificity of interactions between PB1 domains and PCCRs. PB1 domains from p67^phox, PKCζ, Bem1p and scd2 were expressed and purified as GST fusions. On the other hand, PCCRs from p40^phox, ZIP, Cdc24p and scd1 were expressed and purified as MBP fusions. All the possible combinations between these PB1 domains and PCCRs were examined for interactions by in vitro pull-down assays using glutathione–Sepharose.

Fig. 6. Effect of a PB1 domain mutation abolishing the binding of Bem1p to Cdc24p. Left panel: schematic representation of mutants and interactions. Middle panel: the wild-type cells (strain BYM2, sector 1) and bem1^K482A cells (strain BYM1, sector 2) were streaked on an SC-Ura plate and incubated at 37°C for 2 days. Right panel: the bem1^K482A MATa cells (strain BYM1, patch 1) and MATa wild-type cells (strain BYM2, patch 2) were replica-plated to lawns of bem1^K482A MATα cells (strain BYM3) and incubated at 25°C overnight. The plate was then replica-plated to SC-Leu-Ura, on which only diploid cells were able to grow, and incubated for 3 days at 25°C. DH, Dbl-homology domain; PH, pleckstrin-homology domain.

Fig. 7. Domain swapping to prove the roles for the interaction between Bem1p and Cdc24p. (A) Interaction between Bem1p-p67 and Cdc24-p40. Radiolabeled Cdc24p and Cdc24p-p40^phox (left) were incubated with GST–Bem1p or GST–Bem1p-p67^phox. The proteins precipitated with glutathione–Sepharose were subjected to SDS–PAGE and visualized by autoradiography (right). (B) The defects caused by PB1 domain-swapped bem1 are suppressed by the expression of Cdc24p bearing compensatory alterations in the PC motif-containing region. Upper panel: the bem1-p67 cells (strain BYM4) carrying YEpCDC24 (sector 1) or YEpCDC24-p40 (sector 2) and wild-type cells (strain BYM2) carrying a control plasmid (pYO324, a high-copy-number-plasmid with the TRP1 marker; Ohya et al., 1991) (sector 3) were streaked on an SC-Trp-Ura plate and incubated at 37°C for 2 days. Lower panel: patches of the three yeast cells as above were replica-plated to lawns of bem1^K482A MATa cells (strain BYM3) and incubated at 25°C overnight. The plate was then replica-plated to SC-Trp-Leu-Ura, on which only diploid cells were able to grow, and incubated for 3 days at 25°C. (C) Summary of the domain-swapping experiment.