Partitioning-defective Protein 6 (Par-6) Activates Atypical Protein Kinase C (aPKC) by Pseudosubstrate Displacement*

Background: Many cell polarities are controlled by the Par complex, which includes Par-6 and atypical protein kinase C (aPKC). How is aPKC activity controlled? Results: A pseudosubstrate motif within aPKC inhibits its activity. Par-6 displaces the pseudosubstrate and increases aPKC activity. Conclusion: Par-6 activates aPKC. Significance: Par-6 couples aPKC localization and activation to precisely control cell polarity. Atypical protein kinase C (aPKC) controls cell polarity by modulating substrate cortical localization. Aberrant aPKC activity disrupts polarity, yet the mechanisms that control aPKC remain poorly understood. We used a reconstituted system with purified components and a cultured cell cortical displacement assay to investigate aPKC regulation. We find that aPKC is autoinhibited by two domains within its NH2-terminal regulatory half, a pseudosubstrate motif that occupies the kinase active site, and a C1 domain that assists in this process. The Par complex member Par-6, previously thought to inhibit aPKC, is a potent activator of aPKC in our assays. Par-6 and aPKC interact via PB1 domain heterodimerization, and this interaction activates aPKC by displacing the pseudosubstrate, although full activity requires the Par-6 CRIB-PDZ domains. We propose that, along with its previously described roles in controlling aPKC localization, Par-6 allosterically activates aPKC to allow for high spatial and temporal control of substrate phosphorylation and polarization.

The polarization of the cell cortex into molecularly distinct regions underlies remarkably diverse processes, such as junction formation, neurogenesis, and motility. Many cell polarities are orchestrated by the partitioning-defective (Par) 2 complex, which consists of Bazooka (Baz; aka Par-3), Par-6, and atypical protein kinase C (aPKC) (1)(2)(3). Although the Par complex plays a central role in polarizing many cell types, ectopic activity, particularly of aPKC, can lead to loss of polarity with severe consequences such as tissue disorganization and tumorigenesis (4 -6), suggesting that Par complex output must be precisely controlled. In this work, we investigate Par complex regulation by determining the molecular mechanisms by which aPKC activity is controlled.
The Par complex functions to create distinct, polarized cortical domains by coupling phosphorylation to cortical release (7)(8)(9). Through mechanisms that are still being elucidated, the Par complex becomes polarized to one cortical domain and keeps other, cell type-specific factors, localized to an opposite cortical domain (10 -12). The activity of aPKC is a key output of the Par complex because the association of substrates with the cortex can be modulated by phosphorylation (7)(8)(9). For example, in Drosophila neuroblasts the protein Miranda localizes to a cortical domain opposite the Par complex, and its polarization requires aPKC activity (8). Miranda associates with the cortex via its cortical localization domain, but once this domain is phosphorylated by aPKC it is released into the cytoplasm leading to their mutually exclusive localization. Cortical association of the protein Numb is also modulated by aPKC phosphorylation, both in Drosophila and polarized mammalian cells (9), suggesting that coupling of aPKC-mediated phosphorylation to cortical displacement may be a general mechanism for Parmediated polarity.
That activity of aPKC must be maintained within a certain range. In humans, inappropriate aPKC activity is associated with epithelial tumors (5,6). Ectopic aPKC activity in neuroblasts leads to massive overproliferation and concomitant loss of differentiated cells (4). In current models, aPKC activity is controlled by a complex set of protein-protein "scaffolding" interactions (11,13). In particular, Par-6 is thought to repress aPKC (7,14,15), suggesting that aPKC may have a high level of constitutive activity. Par-6 repression of aPKC is thought to be important in Drosophila sensory organ precursor (SOP) cells where aPKC activity is highly dynamic during mitosis (7). Early during SOP division, aPKC is held in an inactive complex along with Par-6 and the tumor suppressor Lethal giant larvae (Lgl). Activation is proposed to occur by phosphorylation of the Par-6 PB1 domain by the mitotic kinase Aurora A. Because the PB1 domain is the interaction site with aPKC (16 -18), Par-6 dissociates from aPKC allowing Lgl to be phosphorylated and released from the complex. Finally, the Par complex member Baz becomes engaged and aPKC becomes fully activated. The mechanism by which aPKC activity is maintained within appropriate levels by dynamic scaffolding interactions has been unclear.
Although protein-protein interactions are thought to regulate aPKC, the precise mechanisms that control catalytic activity are unknown. All PKC isoforms contain NH 2 -terminal domains that are potentially important for controlling the activity of the COOH-terminal kinase domain (19). These domains include the aPKC-specific PB1 that binds Par-6 (18) and the C1 domain that binds lipid cofactors such as diacylglycerol in other PKC family members (20,21), but whose function in aPKCs is unknown (22). PKCs also contain a short pseudosubstrate motif that resembles a true substrate but lacks a phosphorylatable residue, making it capable of acting as a competitive inhibitor. In other PKC isoforms the pseudosubstrate autoinhibits catalytic activity, but its role in regulating aPKC is unclear. In this work, we explore the interplay between internal aPKC regulatory elements and the protein-protein interactions that are thought to control aPKC activity during cell polarization.

EXPERIMENTAL PROCEDURES
Purification of aPKCs and aPKC/Par-6 Complex-HEK293 F cells were transfected with pCMV His 6 -aPKC constructs for expression of individual aPKC variants or co-transfected with pCMV His 6 -Par-6 and pCMV aPKC (no His tag) for expression of the aPKC/Par-6 complex using the 293fectin transfection reagent (Life Technology). The cells were incubated at 37°C for 72 h followed by sonication and centrifugation at 15,000 rpm for 30 min at 4°C. Ammonium sulfate was added to the supernatant to a final concentration of 45% (w/v) and incubated at 4°C for 30 min. The resulting precipitate was collected by centrifugation at 15,000 rpm for 30 min at 4°C and resuspended with Ni 2ϩ lysis buffer (50 mM NaH 3 PO 4 , 300 mM NaCl, 10 mM imidazole, adjusted to pH 8.0 with NaOH). The resuspended precipitate were incubated with Ni 2ϩ -nitrilotriacetic acid resins for 45 min at 4°C. The resins were washed with the lysis buffer. The proteins were eluted using Ni 2ϩ elution buffer (50 mM NaH 3 PO 4 , 300 mM NaCl, 250 mM imidazole, adjusted to pH 8.0 with NaOH). The eluted proteins were dialyzed at 4°C for 4 h. For the complex with Par-6, the proteins were further purified with a size exclusion column (S200 10/30; GE Healthcare). The concentration of aPKC was determined by comparing its reactivity with an anti-aPKC antibody (Santa Cruz Biotechnology) with that of a standard of known concentration (bacterially expressed aPKC kinase domain purified and quantified using a Bradford dye binding assay) on a Western blot.
Expression and Purification of Lgl and Baz-Full-length and residues 647-673 of Drosophila Lgl isoform A and Baz residues 905-1221 of isoform A (constituting the aPKC binding region) were cloned into pMAL-C2 vector (New England BioLabs), in which a tobacco etch virus protease recognition site was added following the MBP coding sequence. The residues Ser 656 and Ser 660 in Lgl 647-673 were mutated to alanines. The constructs were transformed into BL21 Escherichia coli cells. The expressions were induced by 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside at 18°C overnight. The bacterial lysates were incubated with amylose resins (New England BioLabs). The resins were washed with MBP lysis buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM DTT). The MBP fusion proteins were eluted with MBP elution buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 5 mM maltose) and dialyzed at 4°C overnight in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl. The affinity-purified proteins were further purified by ion exchange.
Affinity Chromatography "Pulldown" Binding Assays-GST pulldowns were as described previously (23). Briefly, Drosophila aPKC 120 -141 was cloned into pGEX-4T1 (GE Healthcare) which was transformed into E. coli strain BL21. Protein expression was induced by 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside at 18°C overnight. The bacterial lysate was incubated with GST-agarose (Sigma-Aldrich) at 4°C for 15 min. The GST resins were washed three times with 1ϫ GST pulldown buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.5% Triton X-100). For the experiment in Fig. 1E, 30 g of His-aPKC A134D was added along with 10, 20, 40, 80, or 100 M MBP-Lgl 647-673 S656A/S660A peptide and incubated at room temperature for 20 min in a reaction volume of 100 l. The supernatant was removed and added to amylose resin (New England Biolabs). Both resins were washed three times with 1ϫ GST pulldown buffer. 30 l of 6ϫ SDS loading buffer was added to each sample followed by separation on SDS-PAGE and transfer to nitrocellulose. The membrane was probed for aPKC with rabbit anti-aPKCa (1:5000) (Santa Cruz Biotechnology). The membrane was further incubated with goat anti-rabbit peroxidase-conjugated secondary antibody and visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
In Vitro Kinase Activity Assay-aPKC kinase activity was measured as described previously (24). Briefly, the purified aPKC variants and aPKC/Par-6 complex were diluted to concentrations at which the incorporation of radiolabeled phosphate from [␥-32 P]ATP into MBP-Lgl peptide was linear with respect to time and the enzyme concentrations. The diluted enzymes were preincubated in the assay buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 ) with a wide range of MBP-Lgl peptide concentrations at 30°C for 5 min. The reactions were initiated by adding 1 mM ATP spiked with [␥-32 P]ATP (ϳ1.0 ϫ 10 5 /nmol of ATP). The reactions were incubated at 30°C for 10 min. The reaction mixtures were blotted on Grade P81 phosphopaper (Whatman). The reactions were quenched by immediately submerging the blotted P81 paper in 75 mM H 3 PO 4 . 5 ml of scintillation fluid were added to measure the radioactive decays by liquid scintillation counter. For phosphorylation of Lgl full-length, the reactions were quenched by mixing with 6ϫ SDS loading buffer. The quenched samples were analyzed by 12.5% SDS-PAGE and phosphorimaging. The intensities were analyzed by ImageQuant.
Arg-C Proteinase Sensitivity Assay-Arg-C proteolysis was described previously (25). 30 g of aPKC variants or aPKC/ Par-6 complex was incubated with 1 g of Arg-C proteinase (Sigma-Aldrich) at 37°C for 120 min. Aliquots were removed at 0 and 120 min into equal volume of 6ϫ SDS loading buffer. As negative control, aPKC variants were incubated at the same conditions without Arg-C proteinase. The samples were separated by 8% SDS-PAGE and transferred to nitrocellulose membrane. Western blots were performed to probe for aPKC proteolysis as described above.
S2 Lgl Cortical Localization Assay-Immunofluorescence was as described previously (26). Briefly, for S2 cell expression, aPKC was expressed using transient transfection with a modified pMT vector containing the Drosophila tubulin promoter in place of the metallothionein promoter. Myc:Par-6 and HA:Lgl coding sequences were cloned into the regular pMT vector using 5Ј-BglII and 3Ј-XhoI sites. Drosophila Schneider (S2) cells were maintained in Schneider's medium with 10% FBS at room temperature. ϳ2 ϫ 10 6 cells were seeded/well in a 6-well plate and transfected with 0.5 g of each construct using Effectene transfection reagent according to the manufacturer's protocol. After cells were incubated overnight and induced with 0.5 mM CuSO 4 for 24 h, 200 l of cells were seeded on 12-mm diameter glass coverslips in a 24-well plate and allowed to adhere for 1 h. Cells were fixed for 20 min with 4% formaldehyde in PBS followed by three rinses of wash buffer (0.1% saponin in PBS) and two rinses of block buffer (0.1% saponin and 1% BSA in PBS). Coverslips were incubated overnight at 4°C with the following primary antibodies: rabbit anti-aPKC (1:1000; Santa Cruz Biotechnology), mouse anti-HA (1:1000; Covance), rat anti-Par6 (1:1000; in house). Coverslips were then rinsed three times with blocking buffer, incubated at room temperature for 2 h with species-specific secondary antibodies (1:200; Jackson Immunoresearch), rinsed three times in washing buffer, and mounted in Vectashield Hardset Mounting Medium (Vector Laboratories). Images were acquired on a confocal microscope (Radiance; Bio-Rad Laboratories) using an oil immersion 60ϫ 1.4 NA objective, processed with ImageJ, and assembled in Adobe Illustrator.
Phosphorylation-coupled MBP Pulldown Assay-100 g of the purified MBP-Lgl full-length and MBP-Lgl 647-673 S656A S660A peptide were incubated with amylose resins (New England BioLabs) for 20 min at room temperature. The resins were washed three times with 1ϫ MBP pulldown buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.5% Tween 20) and once with kinase assay buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 ). 30 g of aPKC WT/Par-6 complex was added to the washed resins with or without 0.5 mM ATP to allow phosphorylation. The reaction mixtures were incubated at 30°C for 1 h. The reaction volume was 500 l. The resins were washed three times with 1ϫ MBP pulldown buffer. 6ϫ SDS loading buffer was added to each sample. The samples were separated by 12.5% SDS-PAGE, and binding was analyzed by Western blotting probing for aPKC as described above.

RESULTS
aPKC Is Autoinhibited by Its Pseudosubstrate-We first identified internal elements within aPKC that regulate its kinase activity (aPKC domain structure is shown in Fig. 1A). In current models for aPKC regulation, Par-6 represses kinase activity (7,11,14,15), suggesting that aPKC might be constitutively active. This model originates from experiments using immunoprecipitated aPKC and/or bacterially expressed Par-6 (14,15), in some cases with a nonspecific substrate. To test aPKC regulation rigorously, we used high level expression in HEK293 cells followed by a three-step purification scheme (see "Experimental Procedures"). This method led to variable degrees of purity of the final products, depending on the aPKC variant (a schematic of aPKC variants used in this study is shown in Fig. 1B and supplemental Fig. S1A). Additionally, there was little difference in the degree of activation loop phosphorylation among the variants, with the exception that the K293W ATP binding pocket mutant (27) showed severely reduced modification (Supplemental Fig. S1B). We measured the activity of these preparations in in vitro kinase assays using a peptide from Lgl, a known substrate (28 -30). Lgl contains three phosphorylatable serines, but we mutated two of them to alanine so that only a single site is available for phosphorylation to simplify the analysis (Fig.  1C). We measured initial rates of catalysis by following the transfer of a radiolabeled phosphorus from ATP to the Lgl peptide over a range of substrate concentrations (i.e. a Michaelis-Menten analysis).
We first compared the activities of full-length aPKC with that of its isolated kinase domain. We observed significant levels of activity for the kinase domain, but the activity of full-length aPKC was approximately equal to background activity, as assessed by measuring the activity of the ATP binding pocket mutant K293W (Fig. 1D), which results in an inactive kinase (27). The low activity of full-length aPKC precluded measurement of accurate K m and k cat values for this protein, but analysis of initial rates of the kinase domain yielded values of 4 M and 1.8 s Ϫ1 , respectively. These values are similar to those from other catalytic domains from the AGC family of kinases (31,32). The dramatic difference between the activity of full-length aPKC and its isolated kinase domain suggests that aPKC is autoinhibited and does not require other elements for regulation.
To identify domain(s) that inhibit aPKC activity, we measured the rates of substrate phosphorylation by variants that lacked individual domains within the NH 2 -terminal regulatory region. Like other PKC family members, aPKC contains a pseudosubstrate motif that can act as an internal competitive inhibitor. A peptide containing the pseudosubstrate sequence competes with binding of true substrates (Fig. 1E), suggesting that the pseudosubstrate can interact with the kinase domain active site. This conclusion is further supported by phosphorylation of the pseudosubstrate when the alanine residue that would sit in the active site is mutated to a serine residue (Fig. 1F). Consistent with these observations, deletion of pseudosubstrate domain dramatically increased aPKC activity, indicating that it is required for aPKC autoinhibition (Fig. 1G). To characterize further the role of the pseudosubstrate in autoinhibition, we made versions of aPKC with point mutations in residues thought to be critical for its interaction with the kinase domain. Mutation of the key alanine residue to a phosphomimetic aspartic acid increases kinase activity presumably by causing "substrate release." Additional mutation of basic residues near the phosphorylated residue that are important for kinase domain interaction also increases aPKC activity (Fig. 1G). Thus, the pseudosubstrate is a critical element of aPKC autoinhibition.
We also tested the role of other aPKC domains in regulating catalytic activity. In contrast to the pseudosubstrate, the PB1 domain, which mediates interaction with Par-6, does not directly regulate aPKC activity as its deletion had no detectable effect on catalytic activity (Fig. 2A). The aPKC sequence also contains a C1 domain directly COOH-terminal to the pseudo-substrate. In other PKC isoforms, the C1 binds diacylglycerol, but the C1 function in aPKC is unknown. We found that deletion of C1 increased aPKC activity to a level similar to pseudosubstrate mutants ( Fig. 2A), demonstrating that it plays a critical role in synergizing with the pseudosubstrate to maintain the autoinhibited state.
To dissect further the role of the pseudosubstrate in regulating aPKC, we took advantage of a protease sensitivity assay in which pseudosubstrate cleavage by Arg-C is inhibited by its interaction with the kinase domain (25). The aPKC sequence contains two arginine dipeptides that are located in its pseudosubstrate and could potentially be cleaved by Arg-C (Fig. 1A). However, we observed no cleavage of full-length wild-type aPKC with Arg-C over the course of 120 min (Fig. 2B), consistent with its low catalytic activity. In contrast, the version of aPKC with an aspartic acid mutation in pseudosubstrate domain (A134D), which we found to be highly active in the kinase assay, is Arg-C-sensitive. Mutation of the pseudosubstrate basic residues to alanine in the context of the activating A134D mutation (AADAA) abolished protease sensitivity, confirming that the pseudosubstrate is the cleavage site. Interestingly, we found that removal of the C1 domain increases Arg-C sensitivity of the pseudosubstrate, indicating that the C1 func-tions to regulate aPKC activity by assisting pseudosubstrate interaction with the kinase domain. Thus, deletion of the C1 likely causes a dramatic change in the conformation of aPKC. Taken together, these results indicate that pseudosubstrate protease sensitivity is correlated with catalytic activity, further supporting a model in which aPKC is autoinhibited by the synergistic activity of C1 and pseudosubstrate.
Although our data indicate that aPKC is autoinhibited in vitro, we sought to determine whether it is also autoinhibited in a cellular context. The aPKC substrate Lgl localizes to the cortex of cultured Drosophila S2 cells, but phosphorylation by aPKC causes its displacement into the cytoplasm. When aPKC is expressed with Lgl, a significant fraction remains co-localized at the cortex, consistent with aPKC autoinhibition (Fig. 2, C and  D). When autoinhibition is disrupted, such as when the pseudosubstrate is mutated (leading to high activity in vitro), Lgl is efficiently displaced from the cortex. Thus, both in vitro and in a cellular context, aPKC is autoinhibited.
Par-6 Activates aPKC-Our observation that aPKC is autoinhibited is inconsistent with the current model in which Par-6 inhibits aPKC activity. The lack of detectable kinase activity for full-length aPKC under our assay conditions suggests that further inhibition is unlikely to be physiologically relevant. Thus, LCR, low complexity region. The three residues phosphorylated by aPKC are shown, as is the peptide that was used for the majority of the kinase activity assays showing the phosphorylation sites that were removed by mutation to alanine. C, aPKC constructs used in this study. D, comparison of full-length aPKC catalytic activity with that of the isolated kinase domain. The initial rate of phosphorylation (mol/min) of a MBP fusion of the Lgl peptide (MBP-Lgl peptide; see B) is shown for three aPKC variants: the isolated kinase domain, full-length, and K293W which disrupts the ATP binding site of the kinase domain. E, pseudosubstrate interacting with aPKC and the Lgl peptide competing with their interaction. Addition of MBP-Lgl peptide reduces the amount of aPKC retained on glutathioneagarose beads adsorbed with a GST fusion of the aPKC pseudosubstrate (upper, Western). The supernatant was adsorbed onto amylose resin (lower, Western) to confirm that the MBP-Lgl fusion formed a complex with aPKC. The A134D aPKC pseudosubstrate variant (see B) was used for the pulldown so that the internal pseudosubstrate did not compete with the pulldown. F, pseudosubstrate containing an alanine to serine mutation readily phosphorylated by aPKC. Comparison of the initial phosphorylation rate of a GST fusion of the pseudosubstrate containing mutation A134S (starred residue in A) to MBP-Lgl peptide. The AADAA aPKC pseudosubstrate variant (see B) was used for the kinetic assay. G, activation of aPKC by perturbation of the pseudosubstrate. ⌬PS, aPKC that lacks the pseudosubstrate; A134D, aPKC containing an aspartic acid in place of the starred alanine in A; AADAA, aPKC that contains A134D along with mutations in adjacent basic residues that interact with the kinase domain. The activity of full-length aPKC is shown for comparison. Error bars, S.E.
we decided to revisit the role of Par-6 in aPKC regulation. Previous studies used bacterially expressed Par-6 to investigate its effect on aPKC activity. However, we noticed that bacterially prepared Par-6 is highly aggregated (Fig. 3A), presumably because of its PB1 domain as the CRIB-PDZ fragment is soluble and monomeric. Thus, previously observed aPKC repression may have been due to nonspecific effects of the aggregated protein. To overcome this problem, we co-expressed Par-6 with aPKC in HEK293 cells and purified them together as a complex. When prepared in this manner, Par-6 and aPKC form a discrete complex as assessed by gel filtration chromatography (Fig. 3B) and with high purity as shown by SDS-PAGE (Fig. 3C).
To determine the effect of Par-6 on aPKC activity, we first compared the activity of the co-purified aPKC/Par-6 complex with that of aPKC alone. Par-6 and aPKC interact via their PB1 domains (18), and Par-6 also contains semi-CRIB and PDZ domains (33) (Fig. 4A). Rather than lowering the activity of aPKC, we found that aPKC/Par-6 activity is significantly higher than aPKC alone, comparable with the activity of the aPKC pseudosubstrate mutants (Fig. 4B). This effect is due, in part, to the Par-6 PB1 domain because this domain alone is sufficient to activate aPKC (Fig. 4C). The PB1 does not activate to the same level as full-length Par-6, however, indicating that the CRIB-PDZ domain does participate in aPKC activation. Thus, we conclude that Par-6 activates aPKC through PB1-PB1 interactions and possibly additional interactions elsewhere among the two proteins.
How does Par-6 activate aPKC? To determine whether activation occurs through the pseudosubstrate, we measured the activity of Par-6 in complex with aPKC containing activating pseudosubstrate mutants (Fig. 4D). We observed that this complex has activity similar to the Par-6 complex with wild-type aPKC, suggesting that Par-6 acts through the pseudosubstrate to increase aPKC activity. We verified the effect on the pseudosubstrate by assessing the Arg-C sensitivity of the aPKC/Par-6 complex (Fig. 4E). As opposed to aPKC alone, the pseudosubstrate in the aPKC/Par-6 complex is readily digested by Arg-C. The pseudosubstrate is also accessible to Arg-C in the Par-6 PB1 complex with aPKC, but to a lesser degree, consistent with the lower activity of this complex. We conclude that Par-6 activates aPKC by displacing the pseudosubstrate from the kinase domain.
We also tested whether Par-6 activates aPKC in a cellular context using the Lgl cortical localization assay in S2 cells (Fig.  4, F and G). Whereas aPKC alone is unable to displace Lgl into the cortex, co-expression of Par-6 or just its PB1 domain led to cytoplasmic Lgl, consistent with our in vitro observations. Thus, measurements in vitro and in cells indicate that Par-6 activates aPKC catalytic activity rather than repressing it.
In addition to Par-6, the "Par complex" also includes Baz. As Baz is an aPKC substrate, it may compete with phosphorylation of other substrates like Lgl, although Baz has been proposed to activate aPKC (7). To determine whether Baz influences aPKC activity, we examined the effect of the Baz aPKC binding region (Baz residues 905-1221) on Lgl phosphorylation by Par-6/ aPKC (Fig. 4H). Addition of Baz dramatically decreased the extent of Lgl peptide phosphorylation.
Lgl Is Efficiently Phosphorylated and Released from aPKC/ Par-6 Complex-In Drosophila SOP cells, Lgl is thought to remain in a complex with aPKC until Aurora A activity phos-FIGURE 2. C1 and pseudosubstrate synergistically repress aPKC activity. A, deletion of the PB1 domain has no effect on aPKC catalytic activity whereas loss of the C1 activates aPKC. The activity of the isolated kinase domain and full-length aPKC, both intact and lacking the pseudosubstrate, are shown for comparison. B, mutations that activate aPKC are sensitive to the Arg-C protease. Arg-C protease cleaves at the arginine dipeptides that are located solely in the aPKC pseudosubstrate. Wild-type aPKC is insensitive to Arg-C, presumably because the pseudosubstrate is bound to the kinase domain. The A134D aPKC is Arg-C sensitive, consistent with its increased activity. The AADAA mutation removes the protease cleavage site, confirming that cleavage does not occur at other sites under the conditions used. Deletion of the C1 exposes the pseudosubstrate, indicating that the C1 functions, at least in part, to displace the pseudosubstrate. C, Lgl cortical localization assay for aPKC activity is shown. Lgl localizes to the cortex of cultured S2 cells (first column) even when co-expressed with aPKC (aPKC WT) or a kinase-dead mutant (aPKC K293W) but becomes displaced into the cytoplasm with expressed with an aPKC pseudosubstrate mutant (aPKC RRDRR). Arrowheads indicate cortical Lgl signal. D, Lgl localization in S2 cells was quantified. The percentage of cells with cortical or cytoplasmic Lgl is shown when expressed by itself or with the aPKC variants shown in C. For each condition, 50 cells were examined. Error bars, S.E.   Fig. 1G), has no effect on the aPKC/Par-6 complex. The activities of full-length aPKC and Par-6-activated aPKC are shown for comparison. E, aPKC pseudosubstrate is protease-sensitive when aPKC is bound to full-length Par-6 or its PB1 domain (see Fig. 1G for comparison with wild-type aPKC). F, co-expression of Par-6 or its PB1 domain with aPKC causes cortical displacement of Lgl in S2 cells. Lgl (red signal) associates with the S2 cell cortex, but phosphorylation by aPKC (green) causes displacement into the cytoplasm. Although aPKC itself has no effect on Lgl localization (Fig. 2C), co-expression with Par-6 (blue) leads to cytoplasmic Lgl. G, Lgl localization data shown in F is quantified for 50 cells. H, Bazooka (Baz) inhibits aPKC phosphorylation of the Lgl peptide. Addition of the Baz aPKC binding region (ABR) causes a decrease in the extent of Lgl that is phosphorylated by aPKC. Error bars, S.E.
phorylates Par-6, activating the complex (7). A key prediction of this model is that Lgl is a stable member of a ternary complex with Par-6 and aPKC, even though Lgl is an aPKC substrate. Our work above used a small peptide from Lgl that is phosphorylated by the complex, and it is possible that full-length Lgl represses aPKC activity and remains associated with aPKC/ Par-6. We examined whether aPCK/Par-6 phosphorylated fulllength Lgl in vitro and observed significant phosphorylation, although somewhat less than with the Lgl peptide (Fig. 5A). We also tested whether Lgl is a stable part of the Par complex using a MBP-fused Lgl adsorbed onto amylose resin. This protein efficiently pulls down aPKC/Par-6 in the absence of ATP, but addition of ATP abolished the interaction (Fig. 5B). The result of this interaction assay indicates that MBP-Lgl is released from aPKC/Par-6 once it is phosphorylated, as expected for the substrate of an enzyme, suggesting that Lgl transiently associates with the Par complex.

DISCUSSION
Although aPKC activity is required for many cell polarities, excess activity can lead to tissue disorganization and overproliferation. Thus, a central question in cell polarity is how aPKC catalytic activity is kept in an appropriate range during dynamic processes such as asymmetric cell division. In the current study, we investigated the interplay between elements within aPKC and protein-protein interactions with Par-6 in regulating catalytic activity. We used both an in vitro reconstitution strategy along with a cultured cell cortical displacement assay to measure aPKC activity under a wide variety of contexts. Our observations indicate that aPKC is strongly autoinhibited and that interaction with Par-6 causes activation.
Dual Domain Autoinhibition of aPKC Kinase Domain-We observed that aPKC is strongly autoinhibited. Although the full-length protein has detectable activity, it is significantly less active than the isolated kinase domain. For example, at 10 M substrate, the kinase domain is ϳ50-fold more active than fulllength aPKC. We focused on three NH 2 -terminal domains as candidates for autoinhibition. The PB1 domain that binds the Par complex member Par-6 by PB1 heterodimerization is not required for autoinhibition. Surprisingly, however, aPKC autoinhibition is brought about by collaboration of the two other domains, the kinase-interacting pseudosubstrate and the C1 domain. The aPKC pseudosubstrate efficiently interacts with the kinase domain and is readily phosphorylated when the decoy alanine is replaced with a phosphorylatable residue. Activation of aPKC by mutation of key kinase-interacting residues leads to exposure of the pseudosubstrate, as detected by protease sensitivity. Pseudosubstrate exposure and kinase activation also occur when the C1 domain is deleted, indicating that it plays a previously unappreciated role in regulating kinase activity through the pseudosubstrate. In a recent structure of canonical PKC (34), the C1 domain makes contact with the kinase domain, suggesting that it could make contacts that provide additional stabilization of the pseudosubstrate interaction.
Allosteric Activation of aPKC by Par-6-Although the aPKC PB1 domain is not required for autoinhibition, its interaction with the Par-6 PB1 led to activation. Par-6 activation of aPKC does not lead to full activity, as the kinase domain alone is still significantly more active. However, the ability of aPKC/Par-6 to displace Lgl from the cortex of S2 cells suggests that this level of activity may be sufficient for physiological function. Par-6 activation appears to occur through an allosteric mechanism in which the key inhibitory pseudosubstrate interaction is displaced. How might Par-6 binding influence pseudosubstrate interaction with the kinase domain? Based on the close proximity of the PB1 and pseudosubstrate in the aPKC sequence, we propose a steric model in which PB1-PB1 interaction is incompatible with aPKC autoinhibition (Fig. 5, C and D). In this model the pseudosubstrate occupies the kinase active site and is assisted by C1 interactions with the kinase domain. Recent work on the mammalian form of aPKC also implicates the C1 in regulation (35), although to a greater extent than described here. In canonical PKCs the C1 domain couples diacylglycerol binding to activation by binding to the kinase N-lobe (34,36). In aPKC, activation instead occurs by binding to the PB1 domain which lies on the opposite side of the pseudosubstrate (Fig. 1A).
Par-6 Couples aPKC Localization and Activation-How is aPKC regulated during complex polarization processes such as asymmetric cell division? Current models include Par-6 repression of aPKC as a core component, but our results suggest that these models should be reexamined. How might aPKC autoinhibition and activation by Par-6 regulate polarity? In Drosophila neuroblasts and SOP cells, aPKC is cytoplasmic early in the cell cycle but becomes polarized to the cortex by metaphase. Localization of aPKC occurs by interactions with Par-6, suggesting that this early, unlocalized pool of aPKC may not be  Fig. 1A). B, phosphorylated Lgl is released from the complex. Although MBP-Lgl efficiently pulls down aPKC in the absence of ATP, the addition of ATP causes release of the enzyme, such that it is no longer pulled down. C, model for autoinhibited aPKC shows that the C1 and pseudosubstrate synergistically repress the aPKC catalytic domain. D, model for Par-6-activated aPKC shows that the interaction of the Par-6 PB1 with aPKC causes release of the pseudosubstrate from the aPKC catalytic domain.

Par-6 Activation of aPKC
bound to Par-6 and therefore, autoinhibited. By metaphase, Par-6 becomes polarized to the cortex where it can recruit and activate aPKC. The coupling of aPKC localization to its activation would ensure that substrate phosphorylation only occurs at the correct place and time.
How do other Par complex regulatory components influence activity? The substrates Lgl and Baz have been proposed to regulate aPKC-Lgl by inhibiting and Baz by activating catalytic activity. We have found that Lgl behaves as a typical kinase substrate, only transiently associating with the enzyme. Baz also acts as a substrate, competing for the active site with other substrates such as Lgl. In SOP cells, Lgl is localized uniformly to the cortex early in the cell cycle but ultimately becomes polarized to a domain opposite to aPKC before being completely displaced into the cytoplasm. This dynamic pattern of localization correlates well with low aPKC activity early in the cell cycle (due to autoinhibition) followed by polarized activation from interactions with Par-6. Baz, on the other hand, remains localized in the same cortical domain with Par-6 and aPKC (although it localizes to a separate region in epithelia cells). Further work, both in vitro and in vivo, is required to understand how the constellation of proteins that interact with Par-6 and aPKC regulate catalytic activity and cellular function.