Negative cooperativity underlies dynamic assembly of the Par complex regulators Cdc42 and Par-3
2022; Elsevier BV; Volume: 299; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2022.102749
ISSN1083-351X
AutoresElizabeth Vargas, Kenneth E. Prehoda,
Tópico(s)Heat shock proteins research
ResumoThe Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to the multi-PDZ domain containing protein Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Here, we used a biochemical reconstitution approach with purified, intact Par complex and qualitative binding assays and found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. The energetic coupling arises from interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) that mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity. The Par complex polarizes diverse animal cells through the concerted action of multiple regulators. Binding to the multi-PDZ domain containing protein Par-3 couples the complex to cortical flows that construct the Par membrane domain. Once localized properly, the complex is thought to transition from Par-3 to the Rho GTPase Cdc42 to activate the complex. While this transition is a critical step in Par-mediated polarity, little is known about how it occurs. Here, we used a biochemical reconstitution approach with purified, intact Par complex and qualitative binding assays and found that Par-3 and Cdc42 exhibit strong negative cooperativity for the Par complex. The energetic coupling arises from interactions between the second and third PDZ protein interaction domains of Par-3 and the aPKC Kinase-PBM (PDZ binding motif) that mediate the displacement of Cdc42 from the Par complex. Our results indicate that Par-3, Cdc42, Par-6, and aPKC are the minimal components that are sufficient for this transition to occur and that no external factors are required. Our findings provide the mechanistic framework for understanding a critical step in the regulation of Par complex polarization and activity. Untangling interactions in the PAR cell polarity systemJournal of Biological ChemistryVol. 299Issue 3PreviewAnimal cells establish polarity via the partitioning–defective protein system. Although the core of this system comprises only four proteins, a huge number of reported interactions between these members has made it difficult to understand how the system is organized and functions at the molecular level. In a recent JBC article, the Prehoda group has succeeded in reconstituting some of these interactions in vitro, resulting in a much clearer and simpler picture of partitioning–defective complex assembly. Full-Text PDF Open Access The polarization of animal cells by the Par complex is a highly dynamic, multistep process that begins when actomyosin-generated cortical flows transport membrane-bound Par complex from cellular regions where it is catalytically inactive to a single cortical domain where it becomes activated (1Munro E. Nance J. Priess J.R. Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo.Dev. Cell. 2004; 7: 413-424Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 2Hutterer A. Betschinger J. Petronczki M. Knoblich J.A. Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis.Dev. Cell. 2004; 6: 845-854Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 3Aceto D. Beers M. Kemphues K.J. Interaction of PAR-6 with CDC-42 is required for maintenance but not establishment of PAR asymmetry in C. elegans.Dev. Biol. 2006; 299: 386-397Crossref PubMed Scopus (73) Google Scholar, 4Atwood S.X. Chabu C. Penkert R.R. Doe C.Q. Prehoda K.E. Cdc42 acts downstream of Bazooka to regulate neuroblast polarity through Par-6 aPKC.J. Cell Sci. 2007; 120: 3200-3206Crossref PubMed Scopus (110) Google Scholar, 5Rodriguez J. Peglion F. Martin J. Hubatsch L. Reich J. Hirani N. et al.aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity.Dev. Cell. 2017; 42: 400-415.e9Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Oon C.H. Prehoda K.E. Asymmetric recruitment and actin-dependent cortical flows drive the neuroblast polarity cycle.Elife. 2019; 8e45815Crossref PubMed Scopus (22) Google Scholar, 7Oon C.H. Prehoda K.E. Phases of cortical actomyosin dynamics coupled to the neuroblast polarity cycle.Elife. 2021; 10e66574Crossref PubMed Scopus (7) Google Scholar, 8Lang C.F. Munro E. The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity.Development. 2017; 144: 3405-3416Crossref PubMed Scopus (81) Google Scholar). The transition from an inactive to active complex is mediated by the formation of two distinct complexes: one bound to the multi-PDZ protein Par-3 (Bazooka; Baz in Drosophila) and a Rho GTPase Cdc42-bound complex. Par-3 has many reported interactions with both Par complex components, atypical PKC (aPKC) and Par-6, whereas Cdc42 has one well-defined binding site on Par-6 (Fig. 1A) (9Joberty 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 (773) Google Scholar, 10Lin 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, 11Qiu R.-G. Abo A. Martin G.S. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCζ signaling and cell transformation.Curr. Biol. 2000; 10: 697-707Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Noda 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: human PAR6s link Rac and Cdc42 to aPKC.Genes Cells. 2001; 6: 107-119Crossref PubMed Scopus (116) Google Scholar, 13Garrard 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 (136) Google Scholar, 14Izumi 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 (442) Google Scholar, 15Wodarz 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 (385) Google Scholar, 16Li 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 (29) Google Scholar, 17Renschler 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 (23) Google Scholar, 18Holly 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 (14) Google Scholar, 19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). The transition between these two regulators precisely controls Par complex polarization and activity, with Par-3 coupling the Par complex to cortical flow while inhibiting aPKC activity and GTP-bound Cdc42 maintaining the Par complex at the cell cortex while stimulating aPKC activity (5Rodriguez J. Peglion F. Martin J. Hubatsch L. Reich J. Hirani N. et al.aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity.Dev. Cell. 2017; 42: 400-415.e9Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 20Wang S.-C. Low T.Y.F. Nishimura Y. Gole L. Yu W. Motegi F. Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization.Nat. Cell Biol. 2017; 19: 988-995Crossref PubMed Scopus (73) Google Scholar, 21Dickinson D.J. Schwager F. Pintard L. Gotta M. Goldstein B. A single-cell biochemistry approach reveals PAR complex dynamics during cell polarization.Dev. Cell. 2017; 42: 416-434.e11Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Despite the critical importance of the transition from Par-3 to Cdc42 in the mechanism of Par-mediated polarity, very little is known about how it occurs. While in vivo evidence indicates the Par complex switches from Par-3 to Cdc42-bound states, biochemical evidence suggests that Par-3 and Cdc42 can bind the Par complex simultaneously to form a quaternary complex. A coimmunoprecipitation experiment using cell extracts found that Cdc42-bound Par complexes also contain Par-3 (9Joberty 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 (773) Google Scholar). However, in vivo evidence indicate that there are two distinct cortical pools of Par complex, colocalizing with either Par-3 or Cdc42, and loss of Cdc42 increases the amount of Par-3-bound complex (3Aceto D. Beers M. Kemphues K.J. Interaction of PAR-6 with CDC-42 is required for maintenance but not establishment of PAR asymmetry in C. elegans.Dev. Biol. 2006; 299: 386-397Crossref PubMed Scopus (73) Google Scholar, 5Rodriguez J. Peglion F. Martin J. Hubatsch L. Reich J. Hirani N. et al.aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity.Dev. Cell. 2017; 42: 400-415.e9Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 20Wang S.-C. Low T.Y.F. Nishimura Y. Gole L. Yu W. Motegi F. Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization.Nat. Cell Biol. 2017; 19: 988-995Crossref PubMed Scopus (73) Google Scholar, 22Beers M. Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos.Development. 2006; 133: 3745-3754Crossref PubMed Scopus (45) Google Scholar). The reported ability of Cdc42 and Par-3 to bind simultaneously to the Par complex has influenced models for how the transition between the regulators could occur in vivo. In one model, Cdc42 briefly docks onto Par-3-bound Par complex and activates aPKC, resulting in the phosphorylation and release of Par-3 from the complex (23Morais-de-Sá E. Mirouse V. St Johnston D. aPKC phosphorylation of Bazooka defines the apical/lateral border in Drosophila epithelial cells.Cell. 2010; 141: 509-523Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 24Walther R.F. Pichaud F. Crumbs/DaPKC-dependent apical exclusion of Bazooka promotes photoreceptor polarity Remodeling.Curr. Biol. 2010; 20: 1065-1074Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 25Soriano 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 Scopus (42) Google Scholar). However, recent studies show that phosphorylation of Par-3 by aPKC does not dissociate Par-3 from the Par complex (18Holly 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 (14) Google Scholar, 26Holly 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 (11) Google Scholar). In another proposed model, actomyosin contractility mechanically dissociates Par-3 clusters and facilitates the Par complex transition to Cdc42 (5Rodriguez J. Peglion F. Martin J. Hubatsch L. Reich J. Hirani N. et al.aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity.Dev. Cell. 2017; 42: 400-415.e9Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 20Wang S.-C. Low T.Y.F. Nishimura Y. Gole L. Yu W. Motegi F. Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization.Nat. Cell Biol. 2017; 19: 988-995Crossref PubMed Scopus (73) Google Scholar, 21Dickinson D.J. Schwager F. Pintard L. Gotta M. Goldstein B. A single-cell biochemistry approach reveals PAR complex dynamics during cell polarization.Dev. Cell. 2017; 42: 416-434.e11Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Because the available biochemical data suggest that Par-3 and Cdc42 can bind simultaneously to the Par complex, models for the transition between the two regulators necessarily include other mechanisms (e.g., phosphorylation) or cellular components (e.g., actomyosin contractility). However, the limited in vitro evidence is based on results from cell extracts or experiments using truncated proteins. Additionally, the numerous reported interactions between Par-3 and the Par complex have made it challenging to understand how the Par-3-bound Par complex is regulated. Finally, very little structural information is known about the Par complex and whether the Par-3 and Cdc42 binding sites are in close proximity to one another to regulate the formation of these complexes. Here, we have used a biochemical reconstitution approach with purified components to determine the elements sufficient for Par complex switching between Par-3 and Cdc42. The results provide the mechanistic framework for understanding how the Par complex transitions from Par-3 to Cdc42 to form two distinct complexes. Although Par-3 and Cdc42 are thought to form mutually exclusive complexes with the Par complex in vivo, they have been shown to bind simultaneously in a coimmunoprecipitation experiment using cell extracts (9Joberty 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 (773) Google Scholar). We examined whether Par-3 and Cdc42 influence one another's binding to the Par complex using a reconstitution system. We performed a qualitative affinity chromatography (pull-down) assay with purified Par complex and Par-3 PDZ1-APM (a fragment containing all known interaction motifs between Par-3 and Par-6/aPKC) and GST-fused Cdc42Q61L (constitutively active). The binding buffer included ATP to ensure that the aPKC kinase domain did not form a stalled complex with its phosphorylation site on Par-3. We formed a complex of Cdc42-bound Par-6/aPKC by placing GST-Cdc42Q61L on the solid phase and incubating with soluble, purified Par complex. We assessed the effect of Par-3 on the Cdc42-bound Par complex by adding increasing concentrations of Par-3 PDZ1-APM. If Par-3 binding to the Par complex had no effect on Cdc42 binding or the proteins bound with positive cooperativity, we expected that Par-3 would become part of the solid phase complex and the amount of Par complex adhered to the solid phase would stay the same or increase. Alternately, if the Cdc42 and Par-3 binding sites exhibited negative cooperativity, either via direct steric occlusion or an allosteric mechanism, little or no Par-3 would be part of the Cdc42-bound solid phase complex, and the amount of Par complex on the solid phase would decrease (as the affinity of the Par complex for Cdc42 was reduced by binding to Par-3). We observed that addition of Par-3 significantly reduced the amount of Par complex associated with solid phase Cdc42Q61L (Fig. 1B). Furthermore, little or no additional Par-3 appeared in the solid phase relative to a GST control. Our results indicate that in the context of these four proteins, Cdc42 and Par-3 bind with negative cooperativity to Par-6/aPKC. In a system with two distinct binding sites coupled to one another via negative cooperativity, each protein should reduce the affinity of the Par complex for the other. However, the effect of Cdc42 on Par-3 binding to the Par complex is complicated by the many potential Par-3-binding sites on the Par complex (Fig. 1A). In principle, not all Par-3-binding sites could be coupled to Cdc42 binding, a scenario in which addition of Cdc42 to solid phase Par-3-bound Par complex might not significantly alter the amount of solid phase Par complex. To determine if Cdc42 influences Par-3-bound Par complex, we adsorbed Par complex bound to MBP-Par-3 PDZ1-APM to the solid phase and examined the effect of increasing concentrations of Cdc42Q61L. We observed that addition of Cdc42 reduced the amount of Par complex associated with solid phase Par-3 and Cdc42 was not significantly incorporated into the solid phase (Fig. 1C). Displacement of Par complex from Par-3 required a significantly higher concentration of Cdc42 than we observed for Par-3 displacement of Cdc42-bound complex. Our results indicate that Par-3 and Cdc42 compete for binding to the Par complex (i.e., negative cooperativity) and that a quaternary complex does not form at levels detectable in our assay. Our results may differ from previous studies using cell extracts because aPKC's kinase domain is known to form stalled complexes with substrates like Par-3 when ATP is not available to complete the catalytic cycle (forming a persistent interaction rather than a transient interaction) (18Holly 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 (14) Google Scholar, 26Holly 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 (11) Google Scholar). Additionally, other cellular factors could potentially allow Par-3 and Cdc42 to bind to the Par complex simultaneously. In terms of understanding how the Par complex might transition from Par-3 to Cdc42, our results demonstrate that no other proteins are required—Par-3 and Cdc42 alone are sufficient to form mutually exclusive complexes with the Par complex. Our results indicate that Par-3 is more effective at displacing Cdc42 from the Par complex than Par-3 is at displacing Cdc42. The asymmetry in Par complex displacement could be explained by a higher affinity of Par-3 for the Par complex compared to Cdc42. The affinity of Par-3 PDZ1-APM for the Par complex is known (19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar), but while Cdc42's affinity for the Par-6 CRIB-PDZ fragment has been reported (13Garrard 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 (136) Google Scholar), its affinity for the full Par complex has been unknown. To understand why Par-3 is more effective at displacing Cdc42 from the Par complex, we measured binding affinities for the Par complex using a supernatant depletion assay (27Pollard T.D. A guide to simple and informative binding assays.Mol. Biol. Cell. 2010; 21: 4061-4067Crossref PubMed Scopus (289) Google Scholar). Similar to a previous report using the same assay (19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar), we found that Par-3 PDZ1-APM binds the Par complex with high affinity (Fig. 2; KD of 0.6 μM or ΔG° of 8.3 kcal/mol). We measured a substantially weaker affinity of Cdc42 (using the Q61L constitutively active variant) for the Par complex (Fig. 2; KD of 5.4 μM or ΔG° of 7.1 kcal/mol). This affinity is significantly lower than a previous report of 0.05 μM for Cdc42 binding to the Par-6 CRIB-PDZ fragment using a FRET-based assay (13Garrard 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 (136) Google Scholar). To determine if the source of the difference is Cdc42 binding to a Par-6 fragment versus the full Par complex, we measured the Cdc42 interaction with Par-6 CRIB-PDZ with the supernatant depletion (Fig. S1). While the resulting affinity of 2.3 μM is slightly higher than that for the full Par complex, it remains substantially weaker than the FRET-based value (which is a higher affinity than the Par-3 interaction with the Par complex). We are unsure of the source of this discrepancy, and while our results suggest that Par-3 displaces Cdc42 from the Par complex more efficiently because of an intrinsic difference in affinity, it is possible that there is another source for this phenomenon. Given that Par-3 and Cdc42 bind with negative cooperativity to Par-6/aPKC, we sought to identify the binding sites on the Par complex that are coupled. While the interaction between Cdc42 and Par-6 semi-CRIB is well established, several interactions between Par-3 and the Par complex have been identified (Fig. 1A) (9Joberty 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 (773) Google Scholar, 10Lin 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, 11Qiu R.-G. Abo A. Martin G.S. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCζ signaling and cell transformation.Curr. Biol. 2000; 10: 697-707Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 12Noda 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: human PAR6s link Rac and Cdc42 to aPKC.Genes Cells. 2001; 6: 107-119Crossref PubMed Scopus (116) Google Scholar, 13Garrard 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 (136) Google Scholar, 14Izumi 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 (442) Google Scholar, 15Wodarz 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 (385) Google Scholar, 16Li 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 (29) Google Scholar, 17Renschler 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 (23) Google Scholar, 18Holly 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 (14) Google Scholar, 19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). We excluded the interaction of the aPKC kinase domain with its phosphorylation site on Par-3 (i.e., the Par-3 APM) because the interaction is transient in the presence of ATP, as expected for an enzyme–substrate interaction (18Holly 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 (14) Google Scholar, 26Holly 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 (11) Google Scholar). Given that each Par-3 PDZ domain reportedly interacts with either Par-6 or aPKC, more than one interaction between Par-3 and the Par complex could be involved in displacement of Cdc42 from the Par complex. However, if only one of the interactions between Par-3 and the Par complex displaces Cdc42 from the Par complex, deletion of the required Par-3 element would eliminate Par-3's negative cooperativity with Cdc42 for the Par complex. Alternately, removal of more than one Par-3 element might be necessary to eliminate displacement of Cdc42 from the Par complex by Par-3. To distinguish between these possibilities, we generated deletions of individual Par-3 PDZ domains in the context of the PDZ1-APM fragment and tested which Par-3 elements are involved in displacing Cdc42 from the Par complex. We examined the effect of Par-3 PDZ1-APM, ΔPDZ1, ΔPDZ2, or ΔPDZ3 on Cdc42-bound Par complex. We did not detect an effect of removing PDZ1 or PDZ3 on Par-3's ability to displace Cdc42 from the Par complex (Fig. 3A). In contrast, deletion of Par-3 PDZ2 eliminated displacement of Cdc42 such that the amount of Par complex associated with solid phase Cdc42 did not change upon addition of Par-3 PDZ1-APM ΔPDZ2 (Fig. 3A). Our results indicate that neither Par-3 PDZ1 nor PDZ3 are required for negative cooperativity with Cdc42 for the Par complex and that displacement of Cdc42 from the Par complex by Par-3 is dependent on the PDZ2 domain. We recently discovered that Par-3 PDZ2 and PDZ3 interact with aPKC Kinase Domain-PBM (KD-PBM) module (19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). Given that Par-3 PDZ2 is required to displace Cdc42 from the Par complex, we examined whether the aPKC PBM was also required for this activity. We found that removing the aPKC PBM (Par-6/aPKC ΔPBM) prevented Par-3 from displacing Cdc42 from the Par complex (Fig. 3B). Our results indicate that Par-3 PDZ2 and aPKC PBM are necessary for Par-3 to disrupt the Cdc42–Par complex interaction (Fig. 3C). Given the requirement of the aPKC KD-PBM for Par-3's ability to displace Cdc42 from the Par complex, we examined whether the Par-3 domains that bind the KD-PBM (PDZ2 and PDZ3) were each sufficient for this activity. We recently discovered a conserved basic region (BR) at the N-terminal end of Par-3 PDZ2 that increases PDZ2's affinity for the Par complex, so we also examined the effect of Par-3 BR-PDZ2 (19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). We found that PDZ2 alone was sufficient to displace Cdc42 from the Par complex but was not as effective as PDZ1-APM such that some Par complex remained bound to solid phase Cdc42 (Fig. 4A). In contrast, BR-PDZ2 displaced Cdc42 to a similar extent as PDZ1-APM and resulted in little to no Par complex associated with solid phase Cdc42 (Fig. 4A). We conclude that Par-3 BR-PDZ2 can sufficiently displace Cdc42 from the Par complex. Like Par-3 PDZ2, Par-3 PDZ3 was found to interact with the Par complex utilizing a similar binding mode. Thus, we also tested the ability of Par-3 PDZ3 to displace Cdc42 from the Par complex. Given that Par-3 PDZ3 has a weak binding affinity for the Par complex (KD of 78.9 μM) (19Penkert R.R. Vargas E. Prehoda K.E. Energetic determinants of animal cell polarity regulator Par-3 interaction with the Par complex.J. Biol. Chem. 2022; 298: 102223Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar), we were unable to detect any significant change in the amount of Par complex bound to solid phase Cdc42 (data not shown). Therefore, we instead formed a Par-3 PDZ3-bound Par complex utilizing GST-Par-3 PDZ3 on the solid ph
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