(A–C) Phosphorylation of Par-6 on Ser34 regulates the interaction of Par-6 with aPKC. Recombinant proteins were used to precipitate aPKC from larval brains. (A) The baits were preincubated with recombinant AurA and ATP as indicated. (B) Phosphomimetic forms of Par-6 (S34D/E) and a control mutant (K23A) defective for binding to aPKC (Noda et al., 2003) were used in the pull-down assays. (C) Par-6 interacts with aPKC through dimerization of the PB1 domains.
(D) Phosphorylation on Ser34 is required for full activity of aPKC toward Lgl. Brain extracts from par-6 mutants complemented by genomic rescue constructs expressing either Par-6^WT or Par-6^S34A were analyzed; aurA mutants were included as controls. (#1) and (#2) refer to independent insertions of the rescue construct. Western blots were quantified and normalized for Par-6^WT (set to 1). Averages and standard deviations are shown (n = 3). Differences in p-aPKC and p-Lgl levels between aurA^37/37 and par-6^S34A brains are insignificant (p > 0.45 and p > 0.81, respectively).
(E–N) Phosphorylation on Ser34 is required for timely Numb localization. Larval neuroblasts of the indicated genotypes were stained for p-H3 to label DNA in mitotic cells and for Numb. Apical is up. Percentages shown for prometaphase cells include late prophase cells.

(A and B) Cortical release of Lgl regulates the localization of Baz to the posterior lateral cortex. Baz-GFP was coexpressed with either Lgl-RFP or His-RFP in pupal SOP cells. NEBD is t = 0. Anterior is oriented toward the left. (A) In prophase, Baz-GFP localizes to the posterior lateral cortex, as Lgl-RFP is released from this side. (B) Posterior lateral localization of Baz-GFP fails in aurA^37/37 mutants.
(C and D) AurA promotes and Lgl inhibits the assembly of the Baz complex. Immunoprecipitates (IP) from larval brains expressing Baz-GFP were analyzed. (C’) Quantification of (C). The IP signal was adjusted to the corresponding input signal and normalized for wild-type (WT) (set to 1). Averages and standard deviations are shown (n = 5). Differences to WT are significant (p < 0.05). (D) Immunoprecipitates from brains expressing Baz-GFP alone (control) or together with either Lgl^WT-myc or Lgl^3A-myc were analyzed. (D’) Quantification of (D). The IP signal was adjusted to the corresponding input signal and normalized for control (set to 1). Averages and standard deviations are shown (n = 3 for Baz; n = 6 for Lgl). Baz levels are significantly different from the control, and Lgl levels are significantly different from each other (p < 0.05).

(A) Baz and Lgl assemble into distinct Par complexes with differential activity toward Numb. Baz and Lgl complexes were isolated from baz^CC01941 embryos expressing Baz-GFP by immunoprecipitation (IP) of GFP and Lgl, respectively. The immunoprecipitates were incubated with recombinant Numb, ATP, and aPKC inhibitor where indicated.
(B) Numb is in the Baz complex, but not in the Lgl complex. Recombinant proteins were used in pull-down assays from embryos expressing Baz-GFP.
(C) Numb^S52F is not in the Baz complex. Recombinant proteins were used in pull-down assays from larval brains expressing Baz-GFP.
(D) Proposed mechanism whereby the activation of AurA leads to the phosphorylation of Numb.
(E) Lgl has higher cortical mobility than Numb. Lgl^3A-GFP and Numb-GFP were photobleached on the anterior cortex of pupal SOP cells in metaphase (see Figures S6B and S6C). and the recovery of fluorescence was recorded. The values were normalized to prebleach intensity after correction for background variation and fluorescence loss. Averages are plotted, and standard deviations for the individual time points are shown as gray bars (n = 33 for Lgl^3A-GFP; n = 26 for Numb-GFP).
(F) Numb is dephosphorylated more rapidly than Lgl. myctagged Numb and Lgl were immunoprecipitated from embryos, in vitro phosphorylated by PKCz, and subjected to dephosphorylation by brain extract for the indicated durations. Addition of phosphatase inhibitors suppressed the decay of phosphospecific signal. (F’) Quantification of (F). Averages are plotted, and standard deviations for the individual time points are shown (n = 2).

(A) Lgl is underphosphorylated in aurA mutants. Larval brain extracts of the indicated genotypes were analyzed.
(B) AurA induces the phosphorylation of Lgl by activation of aPKC. Immunoprecipitate (IP) from aurA^37/Ac-3 brains was incubated with ATP and recombinant AurA as indicated.
(C) ClustalW alignment of the AurA phosphorylation site (red) in Par-6 orthologs. Residues identical or similar to the Drosophila protein are colored magenta and blue, respectively. The bottom line shows the AurA consensus (Ferrari et al., 2005); denotes any hydrophobic residue.
(D) Constructs used in the kinase assay.
(E) AurA phosphorylates Par-6 on Ser34 in vitro. Recombinant proteins were incubated with [³²P]ATP and recombinant AurA as indicated. The gel was Coomassie-stained (CBB), followed by autoradiography (³²P). The arrowhead indicates autophosphorylated AurA. Autoradiographs were quantified by summing the signal of the full-length bands and normalizing for the signal of Par-6^WT (set to 1). Averages and standard deviations are shown (n = 4).
(F) AurA phosphorylates Par-6 on Ser34 in vivo. Phosphospecific antibodies directed against Ser34 were used to immunoprecipitate p-Par-6 from brains of the indicated genotypes. A second round (#2) of immunoprecipitation from the supernatant confirms the depletion of p-Par-6. To avoid the overlapping IgG signal and to control for the specificity of the antibodies, extracts from wild-type animals and from par-6^Δ226 mutants complemented by a genomic rescue construct expressing Par-6-GFP (par-6^GFP) were used.

(A–I) Lgl-GFP and Histone-RFP were coexpressed in pupal SOP cells. NEBD is t = 0. Anterior is oriented toward the left. (A) Lgl-GFP is released from the cortex in prophase, is asymmetrically localized (arrowheads) at mid-prophase, and returns to the cortex after mitosis. (B) Expression of aPKC^DN redistributes Lgl-GFP from the cortex into the cytoplasm independent of the cell cycle. (C) Lgl^3A-GFP remains cortical throughout mitosis. (D) Overexpression of Insc inverts Lgl-GFP asymmetry (arrowheads). (E) In aurA^37/37 mutants, cortical release of Lgl-GFP is delayed. (F) Expression of aPKC^ΔN restores cortical release of Lgl-GFP in aurA^37/37 mutants. (G) Lgl-GFP is released from the cortex prematurely when AurA is overexpressed. (H) Lgl^3A-GFP is unaffected by AurA overexpression. (I) Lgl-GFP is released from the cortex in prophase in baz^Xi106 clones.

(A–D) AurA functions in Numb localization by promoting the interaction of Baz with Par-6. GFP-Pon and Histone-RFP were expressed in pupal SOP cells. NEBD is t = 0. The axis of Pon asymmetry (if any) is left to right. Transparent z-projections are shown. (A) In wild-type cells, GFP-Pon is localized asymmetrically from NEBD onward. (B) In aurA mutants, GFP-Pon remains uniformly cortical, but becomes partially polarized in metaphase. (C) Expression of Baz-Par-6 in aurA mutant cells restores the asymmetric localization of GFP-Pon, but does not rescue the delay in mitotic progression. (D) Overexpression of Baz fails to rescue GFP-Pon asymmetry.
(E–M) Larval neuroblasts of the indicated genotypes were stained for p-H3 to label DNA in mitotic cells and for Numb. Apical is up. (E–G) Knockdown of Baz rescues lgl mutants. (H–J) A temperature-sensitive heteroallelic combination of lgl rescues the strong aurA^37/Ac-3 mutant at the restrictive temperature. (K–M) Lgl overexpression enhances the hypomorphic aurA^37/37 mutant.

(A–T) Third-instar larval brains were stained for Miranda and p-H3 (both green) to label neuroblasts and for Prospero (red) to label neurons. Surface views of (A–C) whole brains and (D–T) posterior surface views of single lobes are shown, except in (I), in which both posterior and anterior sides are exposed. Dotted lines demarcate the central brain (CB) (right) from the optic lobe (left). Scale bars are 100 mm. Note that scale varies among the images. (A and D) In the wild-type CB, a defined number of neuroblasts (green) give rise to chains of differentiating progeny (red). (A–G) aPKC regulates neuroblast proliferation through Numb. (B and C) Expression of aPKC^CAAX at 29°C induces strong overproliferation of neuroblasts at the expense of neurons, which is suppressed to wild-type by coexpression of Numb. (E–G) Expression of aPKC^CAAX at 18°C induces modest overproliferation of neuroblasts at the expense of neurons, which is enhanced by heterozygosity for numb¹⁵, but weakly suppressed by heterozygosity for numb^S52F. (H–O) Lgl reg-ulates neuroblast proliferation through Baz and Numb. (H–L) Neuroblast overproliferation in lgl mutants is enhanced by overexpression of Baz-GFP, but suppressed to wild-type by knockdown of Baz. Neither has any effect on neuroblast numbers in the wild-type. (M–O) At the early third-instar stage, lgl mutant neuroblasts weakly overproliferate, which is enhanced by heterozygosity for numb¹⁵, but weakly suppressed by heterozygosity for numb^S52F. (P–T) AurA regulates neuroblast proliferation through subunit exchange in the Par complex. (P–R) Lgl overexpression has no effect on neuroblast numbers in the wild-type, but induces neuroblast overproliferation in aurA^37/37 mutants, which are otherwise free of tumors. (S and T) Baz-GFP overexpression suppresses neuroblast overproliferation in aurA^37/Ac-3 mutants.