Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex
2022; Elsevier BV; Volume: 298; Issue: 8 Linguagem: Inglês
10.1016/j.jbc.2022.102223
ISSN1083-351X
AutoresRhiannon R. Penkert, Elizabeth Vargas, Kenneth E. Prehoda,
Tópico(s)Heat shock proteins research
ResumoThe animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3–Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3–Par complex assembly. The animal cell polarity regulator Par-3 recruits the Par complex (consisting of Par-6 and atypical PKC, aPKC) to specific sites on the cell membrane. Although numerous physical interactions have been reported between Par-3 and the Par complex, it is unclear how each of these interactions contributes to the overall binding. Using a purified, intact Par complex and a quantitative binding assay, here, we found that the energy required for this interaction is provided by the second and third PDZ protein interaction domains of Par-3. We show that both Par-3 PDZ domains bind to the PDZ-binding motif of aPKC in the Par complex, with additional binding energy contributed from the adjacent catalytic domain of aPKC. In addition to highlighting the role of Par-3 PDZ domain interactions with the aPKC kinase domain and PDZ-binding motif in stabilizing Par-3–Par complex assembly, our results indicate that each Par-3 molecule can potentially recruit two Par complexes to the membrane during cell polarization. These results provide new insights into the energetic determinants and structural stoichiometry of the Par-3–Par complex assembly. The Par complex polarizes diverse animal cells by forming a specific domain on the plasma membrane. In the Par domain, the Par complex component atypical PKC (aPKC) phosphorylates and displaces substrates, thereby restricting them to a complementary membrane domain (1Bailey M.J. Prehoda K.E. Establishment of Par-polarized cortical domains via phosphoregulated membrane motifs.Dev. Cell. 2015; 35: 199-210Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this manner, the cellular pattern formed by Par-mediated polarity is ultimately determined by the mechanisms that target the Par complex to the membrane. Membrane recruitment relies at least in part on interactions with proteins that directly associate with the membrane, including the Rho GTPase Cdc42 and the multi-PDZ protein Par-3. The Par complex's interaction with Cdc42 is via a single well-defined site, the Par complex component Par-6's semi-CRIB domain (2Joberty G. Petersen C. Gao L. Macara I.G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42.Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (759) Google Scholar, 3Lin D. Edwards A.S. Fawcett J.P. Mbamalu G. Scott J.D. Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity.Nat. Cell Biol. 2000; 2: 540-547Crossref PubMed Scopus (47) Google Scholar, 4Qiu R.G. Abo A. Steven Martin G. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation.Curr. Biol. 2000; 10: 697-707Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5Noda Y. Takeya R. Ohno S. Naito S. Ito T. Sumimoto H. Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C.Genes Cells. 2001; 6: 107-119Crossref PubMed Scopus (114) Google Scholar, 6Garrard S.M. Capaldo C.T. Gao L. Rosen M.K. Macara I.G. Tomchick D.R. Structure of Cdc42 in a complex with the GTPase-binding domain of the cell polarity protein, Par6.EMBO J. 2003; 22: 1125-1133Crossref PubMed Scopus (130) Google Scholar). However, numerous interactions between Par-3 and the Par complex have been reported (2Joberty G. Petersen C. Gao L. Macara I.G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42.Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (759) Google Scholar, 3Lin D. Edwards A.S. Fawcett J.P. Mbamalu G. Scott J.D. Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity.Nat. Cell Biol. 2000; 2: 540-547Crossref PubMed Scopus (47) Google Scholar, 7Izumi Y. Hirose T. Tamai Y. Hirai S. Nagashima Y. Fujimoto T. et al.An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3.J. Cell Biol. 1998; 143: 95-106Crossref PubMed Scopus (440) Google Scholar, 8Wodarz A. Ramrath A. Grimm A. Knust E. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts.J. Cell Biol. 2000; 150: 1361-1374Crossref PubMed Scopus (376) Google Scholar, 9Renschler F.A. Bruekner S.R. Salomon P.L. Mukherjee A. Kullmann L. Schütz-Stoffregen M.C. et al.Structural basis for the interaction between the cell polarity proteins Par3 and Par6.Sci. Signal. 2018; 11eaam9899Crossref PubMed Scopus (22) Google Scholar, 10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) and it has been unclear how each contributes to the overall interaction. The interaction between Par-3 and the Par complex was originally discovered in the context of the interaction between aPKC and its phosphorylation site on Par-3, the aPKC phosphorylation motif (APM, aka Conserved Region 3–CR3) (7Izumi Y. Hirose T. Tamai Y. Hirai S. Nagashima Y. Fujimoto T. et al.An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3.J. Cell Biol. 1998; 143: 95-106Crossref PubMed Scopus (440) Google Scholar). Subsequently, the following interactions were reported outside of aPKC's catalytic domain: (i) Par-3 PDZ1 and the Par-6 PDZ (2Joberty G. Petersen C. Gao L. Macara I.G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42.Nat. Cell Biol. 2000; 2: 531-539Crossref PubMed Scopus (759) Google Scholar, 3Lin D. Edwards A.S. Fawcett J.P. Mbamalu G. Scott J.D. Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity.Nat. Cell Biol. 2000; 2: 540-547Crossref PubMed Scopus (47) Google Scholar, 11Li J. Kim H. Aceto D.G. Hung J. Aono S. Kemphues K.J. Binding to PKC-3, but not to PAR-3 or to a conventional PDZ domain ligand, is required for PAR-6 function in C. elegans.Dev. Biol. 2010; 340: 88-98Crossref PubMed Scopus (28) Google Scholar), (ii) Par-3 PDZ2-3 acting together and aPKC (8Wodarz A. Ramrath A. Grimm A. Knust E. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts.J. Cell Biol. 2000; 150: 1361-1374Crossref PubMed Scopus (376) Google Scholar), (iii) Par-3 PDZ1 or PDZ3 binding to the Par-6 PDZ-binding motif (PBM) (9Renschler F.A. Bruekner S.R. Salomon P.L. Mukherjee A. Kullmann L. Schütz-Stoffregen M.C. et al.Structural basis for the interaction between the cell polarity proteins Par3 and Par6.Sci. Signal. 2018; 11eaam9899Crossref PubMed Scopus (22) Google Scholar), and (iv) PDZ2 with a PBM in aPKC (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) (Fig. 1A). Each of these interactions, except for the interaction of aPKC's kinase domain with its substrate sequence on Par-3, involves one or more of Par-3's three PDZ protein interaction domains. Several factors have made it difficult to understand how these many interactions identified between Par-3 and the Par complex contribute to the overall interaction. Most interactions have not been examined in the context of the intact Par complex. In this context, it is not possible to understand how individual interactions contribute to the overall energetics of Par-3 assembly with the Par complex or if interactions might cooperate or compete. Furthermore, many of the interactions have not been examined quantitatively, so it has not been possible to assess their relative strength. Finally, the presence of multiple potential Par complex–binding sites on Par-3 raises the possibility that each Par-3 protein might bind more than one Par complex. Here, we examine the energetics of Par-3 binding to the fully reconstituted Par complex using a quantitative binding assay to address these issues. To investigate the energetic determinants of Par-3's interaction with the Par complex (Par-6 and aPKC), we measured binding energy using a supernatant depletion assay (Fig. 1B), using the Drosophila proteins. The supernatant depletion assay uses solid (glutathione or amylose agarose resin) and soluble phases like a typical "pull-down" assay but the amount of protein in the soluble phase ("receptor") is monitored at the equilibrium rather than what remains in the solid phase after washing (12Pollard T.D. A guide to simple and informative binding assays.Mol. Biol. Cell. 2010; 21: 4061-4067Crossref PubMed Scopus (260) Google Scholar) (Fig. 1B and S1A). To confirm that the supernatant depletion assay yields similar affinities to another established protein interaction assay, we measured the affinity of the Crumbs intracellular domain for the Par-6 CRIB-PDZ (6.89 ± 0.07 kcal/mol; mean ± 1 SD, n = 6). This result is indistinguishable from measurements made using the fluorescence anisotropy method (6.89 ± 0.06 kcal/mol) (13Whitney D.S. Peterson F.C. Kittell A.W. Egner J.M. Prehoda K.E. Volkman B.F. Binding of crumbs to the Par-6 CRIB-PDZ module is regulated by Cdc42.Biochemistry. 2016; 55: 1455-1461Crossref PubMed Scopus (20) Google Scholar). For measuring Par complex affinities for Par-3, we used the PDZ1-APM region of Par-3 (Fig. 1A) as a starting point because it contains all domains that have been reported to interact with the Par complex and it can be purified to a level suitable for quantitative analysis (all protein reagents used in this study are shown in Fig. S1B) (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). We examined the binding of Par-3 PDZ1-APM to the full Par complex to allow the multiple, potentially cooperative interactions to form. As shown in Fig. 1, C and D, the binding energy (ΔG°) of Par-3 PDZ1-APM to the Par complex is 9.1 kcal/mol (9.0–9.2 95% CI; all binding energies reported in this study can be found in Table S1). Because of the potential for multiple interactions between Par-3 and the Par complex, this energy may be the cumulative effect of individual binding events. For this reason, binding energies for reactions that have stoichiometries that are potentially greater than one are labeled as "apparent". Below, we examine how each of the potential interactions between Par-3 and the Par complex contributes to the overall binding energy. We recently discovered an interaction between the second of Par-3's three PDZ domains (PDZ2) and a highly conserved PBM at the COOH-terminus of aPKC (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The Par-3 PDZ2–aPKC PBM interaction is required for the recruitment of the Par complex to the cortex of asymmetrically dividing Drosophila neural stem cells. Using the supernatant depletion assay, we found that this interaction has an apparent binding energy of 5.5 kcal/mol (5.3–5.7 95% CI), which represents approximately 60% of the full Par-3 PDZ1-APM's binding energy and is indistinguishable from PDZ1-APM binding to the aPKC PBM (Fig. 1, C and D). We were unable to detect an interaction between PDZ2 and a Par complex lacking aPKC's PBM (the limit of detection of the supernatant depletion assay is approximately 4.5 kcal/mol) consistent with a central role for this motif in the overall interaction. Surprisingly, however, removal of PDZ2 did not abrogate binding as PDZ1-APMΔPDZ2 bound the Par complex with approximately the same binding energy as that for Par-3 PDZ2–aPKC PBM (Fig. 1, C and D; 5.6 kcal/mol; 5.6–5.7 95% CI). We conclude that while Par-3 PDZ2–aPKC PBM represents a significant fraction of the Par-3 interaction with the Par complex, interactions outside of the PDZ2 (but also potentially involving the aPKC PBM) make a significant contribution. Furthermore, individual interactions appear to be nonadditive (i.e., cooperative). We sought to determine which interaction domains or motifs from the Par complex collaborate with the aPKC PBM to contribute binding energy for Par-3. Par-6 has been reported to contain a PBM that interacts with Par-3 PDZ1 or PDZ3 (9Renschler F.A. Bruekner S.R. Salomon P.L. Mukherjee A. Kullmann L. Schütz-Stoffregen M.C. et al.Structural basis for the interaction between the cell polarity proteins Par3 and Par6.Sci. Signal. 2018; 11eaam9899Crossref PubMed Scopus (22) Google Scholar). When examining the effect of removing Par-6's PBM on the overall interaction energetics, we were unable to detect a difference in the binding of Par-3 to the Par complex lacking the Par-6 PBM (Fig. 2, A and B; 9.3 kcal/mol; 8.9–9.6 95% CI). Given that our implementation of the supernatant depletion assay reliably detects binding energy differences on the order of 0.2 kcal/mol (e.g., see confidence intervals in Fig. 1C) and that we were unable to detect an interaction between Par-3 PDZ1-APM and Par-6ΔPB1 (Fig. 2, A and B), we conclude that Par-3 interactions with the Par-6 PBM do not play a significant role in stabilizing Par-3 binding to the Par complex in the context of these purified components. Given that the Par-6 PBM is not responsible for the additional interaction energy with Par-3, we sought to determine which Par complex interaction domains or motifs might contribute to the additional binding energy beyond the aPKC PBM. We found that the aPKC kinase domain along with the adjacent PBM (KD-PBM; Fig. 2C) fully recapitulated the interaction energy of the Par complex with Par-3 (Fig. 2, A and B; 9.0 kcal/mol; 8.8–9.2). Thus, in the context of these purified components, we do not find that the Par-3 PDZ1 interaction with the Par-6 PDZ or the PDZ1 and 3 interactions with the Par-6 PBM substantially contribute to the overall Par-3 and Par complex–binding energy. We used both the Par complex and the isolated aPKC KD-PBM to identify which regions of Par-3 outside of PDZ2 contribute to the overall interaction energy. We found that a Par-3 fragment containing its three PDZ domains has a similar binding energy as PDZ1-APM (Fig. 3, A–C; 9.3 kcal/mol; 9.0–9.6 95% CI). This result indicates that Par-3's phosphorylation site (APM) and the linker region connecting it to PDZ3 do not contribute significantly to the interaction. Note that ATP was present in our binding assay so that any interaction of the aPKC kinase domain with the APM was likely transient (and the interaction with the phosphorylated APM is weak) (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 14Holly R.W. Prehoda K.E. Phosphorylation of Par-3 by atypical protein kinase C and competition between its substrates.Dev. Cell. 2019; 49: 678-679Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). We did not detect any difference in the binding energy of Par-3 PDZ1-3 to the Par complex when ATP was replaced with ADP (Fig. S2A). When examining the three Par-3 PDZ domains, we found that either PDZ1-2 or PDZ2-3 bound with binding energies similar to PDZ1-APM, although PDZ1-2's was somewhat lower than PDZ2-3's, an effect that was larger in the context of the full Par complex relative to the KD-PBM alone (Fig. 3). We noticed that an approximately 30 residue sequence NH2-terminal to the PDZ2 domain is enriched in basic amino acids and highly conserved in Par-3's from diverse animal species (Fig. 4A). We termed this motif the basic region (BR) and found that including it with Par-3 PDZ2 (BR-PDZ2) significantly increased the binding energy of the interaction with the aPKC kinase domain and the full Par complex (Fig. 4, B and C). We conclude that the higher binding energy of PDZ1-2 than PDZ2 alone is contributed by the conserved BR motif. Like PDZ1-2, the combination of PDZ2 and 3 (Par-3 PDZ2-3) also bound aPKC KD-PBM and the full Par complex with higher affinity than PDZ2 alone (Fig. 5, A–D). In this case, the higher binding energy originates from PDZ3 as we discovered that it binds the aPKC KD-PBM with similar energy to PDZ2 and with somewhat less energy to the full Par complex (Fig. 5, A–D). We also found that PDZ3 binds the aPKC PBM with a similar energy as PDZ2. Like PDZ2, the binding energy of PDZ3 was higher for aPKC KD-PBM than the PBM alone. Our results indicate that Par-3 PDZ2 and PDZ3 use a similar binding mode and therefore may compete for binding to aPKC KD-PBM. To test this hypothesis, we performed a competition experiment, first assembling a complex of the aPKC KD-PBM with PDZ2 and then adding PDZ3. We found that the presence of PDZ3 caused a significant decrease in the amount of aPKC bound to PDZ2 (Fig. 5E). Soluble PDZ2 was also able to displace PDZ3 from aPKC KD-PBM (Fig. 5E). The competitive binding for the two PDZ domains suggests that PDZ2 and PDZ3 each binds a distinct Par complex. Furthermore, the increased binding affinity when both PDZ2 and 3 are present (e.g., PDZ2-3) relative to the individual domains likely arises from an avidity effect in which more than one Par complex is participating in the interaction. Taken together, our results suggest that the binding energy of the Par-3 interaction with the Par complex arises from separate interactions of the BR-PDZ2 and PDZ3 with the aPKC KD-PBM. As shown in Figure 6, A and B, Par-3 BR-PDZ2-3 nearly completely recapitulates the binding energy of PDZ1-APM. To determine if distinct Par complexes can bind to PDZ2 and PDZ3, we asked whether the Par-3 PDZ1-APM adsorbed to the solid phase via the aPKC PBM could recruit the Par complex. We found that the Par complex was specifically adsorbed to GST-aPKC PBM in the presence of Par-3 PDZ1-APM (Fig. 6C). We conclude that distinct interactions of BR-PDZ2 and PDZ3 with aPKC KD-PBM form the basis of the Par-3 interaction with the Par complex (Fig. 6D). In the context of these purified proteins, we do not detect a significant contribution from the interaction of PDZ1 or PDZ3 with the Par-6 PBM, the interaction of PDZ1 with the Par-6 PDZ, or the interaction of the aPKC kinase domain with its phosphorylation site. The nature of the Par-3 interaction with the Par complex has been enigmatic (15Lang C.F. Munro E. The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity.Development. 2017; 144: 3405-3416Crossref PubMed Scopus (67) Google Scholar, 16Wu X. Cai Q. Feng Z. Zhang M. Liquid-liquid phase separation in neuronal development and synaptic signaling.Dev. Cell. 2020; 55: 18-29Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 17Riga A. Castiglioni V.G. Boxem M. New insights into apical-basal polarization in epithelia.Curr. Opin. Cell Biol. 2020; 62: 1-8Crossref PubMed Scopus (29) Google Scholar, 18Thompson B.J. Par-3 family proteins in cell polarity & adhesion.FEBS J. 2022; 289: 596-613Crossref PubMed Scopus (9) Google Scholar, 19Martin E. Girardello R. Dittmar G. Ludwig A. New insights into the organization and regulation of the apical polarity network in mammalian epithelial cells.FEBS J. 2021; 288: 7073-7095Crossref PubMed Scopus (13) Google Scholar). In this study, we used a quantitative biochemical approach with purified, full-length Par complex and a region of Par-3 that contains all known binding motifs to address the challenge of understanding this complicated interaction. We found that Par-3 PDZ2 and PDZ3 binding to the aPKC KD-PBM nearly fully recapitulates the binding energy of the overall interaction between Par-3 and the Par complex. We note that these interactions most closely resemble the previously identified interaction of Par-3 PDZ2-3 with full-length aPKC using a yeast two-hybrid assay (8Wodarz A. Ramrath A. Grimm A. Knust E. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts.J. Cell Biol. 2000; 150: 1361-1374Crossref PubMed Scopus (376) Google Scholar). We used the Drosophila versions of these proteins and, while the Par complex is highly conserved, it is possible that the proteins from other organisms behave differently. Here, we examine the implications of our quantitative findings on Par-3's role in Par-mediated polarity. We used binding energy to evaluate the relative contribution of each of the identified Par-3 interactions with the Par complex. The binding energies of several of the interactions in the context of isolated Par complex fragments have been previously reported. The interaction of the aPKC kinase domain with the Par-3 APM has been reported to be very high (8.6 kcal/mol) (20Soriano E.V. Ivanova M.E. Fletcher G. Riou P. Knowles P.P. Barnouin K. et al.aPKC inhibition by Par3 CR3 flanking regions controls substrate access and underpins apical-junctional polarization.Dev. Cell. 2016; 38: 384-398Abstract Full Text Full Text PDF PubMed Google Scholar). However, this interaction was measured in the absence of ATP, conditions which prevent substrate turnover and are consequently not physiologically relevant (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 14Holly R.W. Prehoda K.E. Phosphorylation of Par-3 by atypical protein kinase C and competition between its substrates.Dev. Cell. 2019; 49: 678-679Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). We did not detect any contribution to the overall interaction between Par-3 and the Par complex from the Par-3 APM when ATP was present. The interactions of Par-3 PDZ1 and PDZ3 with the Par-6 PBM were measured using NMR and were found to be weak (5.0 and 5.8 kcal/mol, respectively). While these affinities are low, they are above the limit of detection of the supernatant depletion assay. However, we did not detect any significant contributions from the Par-6 PBM in the context of the Par complex binding to Par-3; we did not detect an interaction of Par-3 with Par-6/aPKCΔPBM nor did we detect a change in affinity when the Par-6 PBM was removed (i.e., Par-6ΔPBM/aPKC). An analysis of the Par-3 domains required for polarity in the Caenorhabditis elegans zygote found that PDZ1 and 3 were dispensable but the oligomerization domain and PDZ2 were necessary (21Li B. Kim H. Beers M. Kemphues K. Different domains of C. elegans PAR-3 are required at different times in development.Dev. Biol. 2010; 344: 745-757Crossref PubMed Scopus (34) Google Scholar). A similar analysis found that the interaction of the Par-6 PDZ domain with Par-3 was also dispensable for the Par complex function (11Li J. Kim H. Aceto D.G. Hung J. Aono S. Kemphues K.J. Binding to PKC-3, but not to PAR-3 or to a conventional PDZ domain ligand, is required for PAR-6 function in C. elegans.Dev. Biol. 2010; 340: 88-98Crossref PubMed Scopus (28) Google Scholar). An examination of the Par-6 PBM found that it is not required for viability in Drosophila and its removal did not have a measurable effect on Par-6 recruitment to the cortex of the embryonic epithelium except when the Par-6 PDZ was also removed (9Renschler F.A. Bruekner S.R. Salomon P.L. Mukherjee A. Kullmann L. Schütz-Stoffregen M.C. et al.Structural basis for the interaction between the cell polarity proteins Par3 and Par6.Sci. Signal. 2018; 11eaam9899Crossref PubMed Scopus (22) Google Scholar). In a study of aPKC PBM function, aPKCΔPBM was not polarized to the apical membrane during the asymmetric division of Drosophila larval neural stem cells (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). These functional results are consistent with the primacy of the aPKC PBM in binding to Par-3. They also suggest that the biochemical redundancy between Par-3 PDZ2 and PDZ3 does not translate to in vivo function, either because of the lower affinity of PDZ3 or because PDZ2 participates in other essential functions besides binding to the Par complex. Our results indicate that the aPKC kinase domain participates in the interaction with Par-3 PDZ2 and PDZ3. The nature of this interaction is not known, but the proximity of the aPKC PBM to the kinase domain is suggestive (Fig. 2C) (20Soriano E.V. Ivanova M.E. Fletcher G. Riou P. Knowles P.P. Barnouin K. et al.aPKC inhibition by Par3 CR3 flanking regions controls substrate access and underpins apical-junctional polarization.Dev. Cell. 2016; 38: 384-398Abstract Full Text Full Text PDF PubMed Google Scholar). Binding to the PBM would bring PDZ surfaces outside of the PBM pocket near the kinase domain and could lead to the so-called "docking" interactions that occur between protein kinase substrates and regions away for the kinase domain active site (22Reményi A. Good M.C. Lim W.A. Docking interactions in protein kinase and phosphatase networks.Curr. Opin. Struct. Biol. 2006; 16: 676-685Crossref PubMed Scopus (145) Google Scholar). Another interesting feature of the binding energetics results is the higher binding energy of the Par-3 PDZ domains to the aPKC KD-PBM than the full Par complex (Fig. 5A versus Fig. 5B). The lower binding affinity to the full Par complex suggests that autoregulation may be present in the system. Future efforts will be directed at exploring the nature of these interactions and any role they may have in regulating aPKC activity. GST-, MBP-, and his-tagged Par-3 constructs, GST-aPKC PBM, and his aPKC kinase domain-PBM (residues 259–606) were cloned as previously described (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) using Gibson cloning (New England Biolabs), Q5 mutagenesis (New England Biolabs), or traditional methods. In addition to an N-terminal MBP tag, the Par-3 PDZ1-APM (residues 309–987) construct also contained a C-terminal his-tag. Par complex components (aPKC and his-Par-6) were cloned into pCMV (human cytomegalovirus) as previously described (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 23Graybill C. Wee B. Atwood S.X. Prehoda K.E. Partitioning-defective protein 6 (Par-6) activates atypical protein kinase C (aPKC) by pseudosubstrate displacement.J. Biol. Chem. 2012; 287: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Please see the Key resources table for additional information on specific constructs. All proteins, except for Par complex constructs, were expressed in Escherichia coli (strain BL21 DE3). Constructs were transformed into BL21 cells, grown overnight at 37 °C on LB+ampicillin (Amp; 100 μg/ml). Resulting colonies were selected and used to inoculate 100 ml LB+Amp starter cultures. Cultures were grown at 37 °C to an A600 of 0.6 to 1.0 and then diluted into 2 l LB+Amp cultures. At an A600 of 0.8 to 1.0, expression was induced with 0.5 mM IPTG for 2 to 3 h. Cultures were centrifuged at 4400g for 15 min to pellet cells. The media was removed and pellets were resuspended in nickel lysis buffer [50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, pH 8.0], GST lysis buffer [1XPBS, 1 mM DTT, pH 7.5] or maltose lysis buffer [20 mM Tris, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, and pH 7.5], as appropriate. Resuspended pellets were frozen in liquid N2 and stored at −80 °C. Par complex constructs were expressed in HEK 293F cells (Thermo Fisher Scientific), as previously described (10Holly R.W. Jones K. Prehoda K.E. A conserved PDZ-binding motif in aPKC interacts with Par-3 and mediates cortical polarity.Curr. Biol. 2020; 30: 893-898.e5Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 23Graybill C. Wee B. Atwood S.X. Prehoda K.E. Partitioning-defective protein 6 (Par-6) activates atypical protein kinase C (aPKC) by pseudosubstrate displacement.J. Biol. Chem. 2012; 287: 21003-21011Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, cells were grown
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