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. 2004 Aug 16;166(4):549–557. doi: 10.1083/jcb.200311031

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Copyright © 2004, The Rockefeller University Press

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Figure 3. — Crb is a phosphorylation target of DaPKC. (A) In vitro kinase assay. The indicated GST-fusion proteins were subjected to aPKCζ phosphorylation in vitro and resolved by SDS-PAGE followed by autoradiography. Arrow points to phosphorylated Crbi. Asterisk marks autophosphorylated aPKC. Patj (position indicated by arrowhead) is not phosphorylated. Note that the presence of Patj largely prevents Crbi phosphorylation. Position of molecular weight markers is indicated on the left. Immunoblotting with anti-aPKC is shown in the bottom left panel. (Right) Staining with Coomassie blue of the total protein loaded. (B) Alignment of the amino acid sequences of the cytoplasmic domains of Drosophila (DmCrbi), mouse Crb3 (MmCrbi3), human Crb3 (HCrbi3), and C. elegans Crb1 (CeCrbi1). The conserved Thr and Ser residues (T6, T9, S11, and S13) are indicated with asterisks. All of these amino acids were mutated to A in the Crbi^{T6AT9AS11AS13A} protein. T6 and T9 were mutated to D in DmCrbi^T6DT9D and to A in DmCrbi^T6AT9A. (C) Mutagenesis analysis of the phosphorylation sites of Crbi. aPKC-dependent in vitro phosphorylation was performed with the indicated fusion proteins. aPKC is unable to phosphorylate the mutated Crbi devoid of its four Ser/Thr residues (top left, Crbi^{T6AT9AS11AS13A}). Phosphorylation is restored only after reversion in the mutated Crbi protein of Ala6 or Ala9 to Thr (top middle, named ABT6 and ABT9) Mutated Crbi^T6AT9A is not phosphorylated by aPKC (top right). Total proteins, stained with Coomassie blue, are shown in the bottom panels as loading control.