A PDZ-kinase allosteric relay mediates Par complex regulator exchange

Abstract

The Par complex polarizes the plasma membrane of diverse animal cells using the catalytic activity of atypical PKC (aPKC) to pattern substrates. Two upstream regulators of the Par complex, Cdc42 and Par-3, bind separately to the complex to influence its activity in different ways. Each regulator binds a distinct member of the complex, Cdc42 to Par-6 and Par-3 to aPKC, making it unclear how they influence one another's binding. Here, we report the discovery that Par-3 binding to aPKC is regulated by aPKC autoinhibition and link this regulation to Cdc42 and Par-3 exchange. The Par-6 PDZ domain activates aPKC binding to Par-3 via a novel interaction with the aPKC kinase domain. Cdc42 and Par-3 have opposite effects on the Par-6 PDZ-aPKC kinase interaction: while the Par-6 kinase domain interaction competes with Cdc42 binding to the complex, Par-3 binding is enhanced by the interaction. The differential effect of Par-3 and Cdc42 on the Par-6 PDZ interaction with the aPKC kinase domain forms an allosteric relay that connects their binding sites and is responsible for the negative cooperativity that underlies Par complex polarization and activity.

Keywords: PDZ domain; Par complex; allostery; cell polarity; protein kinase.

Figure 1

Par-3 binding to aPKC is autoinhibited by the regulatory domain of aPKC.A, schematic of the domain architecture of and interactions between the Par complex proteins (aPKC and Par-6) and known regulators Par-3 (Bazooka in Drosophila) and Cdc42. B, Par-3 binding to aPKC kinase domain with its associated PDZ-binding motif (KD-PBM) or full-length aPKC (aPKC FL). Solid phase (glutathione resin)-bound glutathione-S-transferase (GST) or GST-fused Par-3 PDZ2 with its associated basic region (BR-PDZ2) incubated with aPKC KD-PBM or full length (aPKC FL). Shaded regions indicate the fraction applied to the gel (soluble phase or solid phase components after incubation with indicated soluble components and washing). C, Par-3 binding to aPKC KD-PBM and/or regulatory domain (aPKC PB1-C1). Labeling as described in (B). D, Gardner-Altman estimation plot of normalized band intensity of aPKC KD-PBM binding to Par-3 BR-PDZ2 in the presence or absence of its regulatory domain (aPKC PB1-C1). The results of each replicate (filled circles) are shown along with mean and standard deviation (gap and bars adjacent to filled circles). The mean difference is plotted on the right as a bootstrap sampling distribution (shaded region) with a 95% confidence interval (black error bar). E, summary of Par-3 interactions with aPKC. aPKC, atypical PKC.

Figure 2

Par-3 binding to aPKC is activated by Par-6.A, Par-3 binding to aPKC in the presence or absence of various Par-6 domains. Solid phase (glutathione resin)-bound glutathione-S-transferase (GST)-fused Par-3 PDZ2 with its associated basic region (BR-PDZ2) incubated with full-length aPKC (aPKC FL), Par complex (full-length aPKC and Par-6), Par complex ΔCRIB-PDZ (aPKC with the Par-6 PB1 domain), or Par complex ΔCRIB-PDZ plus Par-6 CRIB-PDZ. Shaded regions indicate the fraction applied to the gel (soluble phase or solid phase components after incubation with indicated soluble components and washing). Insets shows enlargement of the last four lanes (top-to more clearly show aPKC FL and contaminant band) or last five lanes (bottom-with contrast increased to show faint Par-6 CRIB-PDZ and PB1 bands). B, Cumming estimation plot of the normalized band intensity of aPKC binding to Par-3 BR-PDZ2 under the indicated conditions shown in (A). The result of each replicate (filled circles) along with the mean and SD (gap and bars next to circles) are plotted in the top panel and the mean differences are plotted in the bottom panel as a bootstrap sampling distribution (shaded region) with a 95% confidence interval (black error bar). C, effect of Par-6 CRIB-PDZ on the affinity of the Par complex for Par-3. Cumming estimation plot of Par-3/Par complex interaction energies measured using the supernatant depletion assay. The result of each replicate is shown (filled circles) along with mean and SD (gap and bars adjacent to filled circles). The difference in the means is shown along with the 95% confidence intervals (black bar) derived from the bootstrap 95% confidence interval (shaded distribution). D, summary of Par-3 interactions with Par complex. aPKC, atypical PKC.

Figure 3

Par-6 PDZ binds directly to the kinase domain of aPKC.A, Par-6 CRIB-PDZ binding to the various domains of aPKC. Solid phase (glutathione resin)-bound glutathione-S-transferase (GST)-fused Par-6 CRIB-PDZ incubated with the regulatory module of aPKC (PB1-C1) or aPKC kinase domain (KD) with its PDZ-binding motif (PBM). Shaded regions indicate the fraction applied to the gel (soluble phase or solid phase components after incubation with indicated soluble components and washing). B, binding of individual domains of Par-6 to aPKC KD or KD-PBM. Solid phase (glutathione resin)-bound glutathione-S-transferase (GST)-fused Par-6 CRIB-PDZ, GST-fused Par-6 CRIB, or GST-fused Par-6 PDZ incubated with either aPKC KD-PBM or aPKC KD only. Shaded regions indicate the fraction applied to the gel (soluble phase or solid phase components after incubation with indicated soluble components and washing). aPKC, atypical PKC; PBM, PDZ-binding motif.

Figure 4

Par-6 CRIB-PDZ and Par-3 PDZ2 bind cooperatively to aPKC.A, binding of Par-6 CRIB-PDZ to aPKC in the presence or absence of Par-3. Solid phase (glutathione resin)-bound glutathione-S-transferase (GST)-fused Par-6 CRIB-PDZ incubated with Par complex ΔCRIB-PDZ in the presence or absence of Par-3 PDZ2 and its associated basic region (BR-PDZ2). Shaded regions indicate the fraction applied to the gel (soluble phase or solid phase components after incubation with indicated soluble components and washing). B, Gardner-Altman estimation plot of normalized band intensity of aPKC binding to Par-6 CRIB-PDZ in the absence or presence of Par-3 BR-PDZ2. C, summary of Par-6 CRIB-PDZ and Par-3 BR-PDZ2 cooperative binding to aPKC. aPKC, atypical PKC.

Figure 5

Cdc42 displaces Par-6 CRIB-PDZ from the aPKC kinase domain.A, binding of Par-6 CRIB-PDZ to Cdc42 and aPKC KD with its PDZ-binding motif (KD-PBM). Solid phase (glutathione resin)-bound glutathione-S-transferase (GST)-fused Par-6 CRIB-PDZ incubated with Cdc42 Q61L, aPKC KD-PBM, or both Cdc42 and aPKC KD-PBM. B, Gardner-Altman estimation plot of normalized band intensity of aPKC KD-PBM binding to Par-6 CRIB-PDZ in the absence or presence of Cdc42 Q61L. C, summary of Cdc42’s effect on the Par-6 CRIB-PDZ interaction with the aPKC KD. aPKC, atypical PKC.

Figure 6

Cdc42 toggles the interaction between Par-6 CRIB-PDZ and aPKC kinase domain to influence Par-3 binding.A, binding of Par-3 to aPKC in the presence of Par-6 CRIB-PDZ with or without Cdc42. Solid phase (glutathione resin)-bound glutathione-S-transferase (GST)-fused Par-3 PDZ2 with its associated basic region (BR-PDZ2) incubated with Par complex (full-length aPKC and Par-6) or Par complex ΔCRIB-PDZ (full-length aPKC plus the Par-6 PB1 domain) with Par-6 CRIB-PDZ and Cdc42Q61L as indicated. Shaded regions indicate the fraction applied to the gel (soluble phase or solid phase components after incubation with indicated soluble components and washing). Inset shows enlargement of the last four lanes. B, Cumming estimation plot of normalized band intensity of aPKC binding to Par-3 BR-PDZ2 under the indicated conditions from (A). The result of each replicate (filled circles) along with the mean and SD (gap and bars next to circles) are plotted in the top panel and the mean differences are plotted in the bottom panel as a bootstrap sampling distribution (shaded region) with a 95% confidence interval (black error bar). C, summary of PDZ-kinase mediated regulator exchange. Par-3 BR-PDZ2 binds with low affinity to aPKC because of autoinhibition. Par-6 relieves autoinhibition through its PDZ domain interacting with the aPKC kinase domain leading to high affinity Par-3 binding. Cdc42 inhibits the PDZ–kinase interaction to restore aPKC to its low Par-3 affinity state. aPKC, atypical PKC.