FIGURE 5.

CUX1 is phosphorylated at several SP dipeptide sites in mitosis. A, diagrammatic representation of the recombinant CUX1(612–1328/HA) protein showing the phosphorylation sites for PKA (Ser¹²¹⁵ and Ser¹²¹⁶), CKII (Ser⁹⁷⁷ and Ser¹¹⁶⁰), PKC (Thr⁹⁹² and Ser¹¹⁷⁵), and the SP dipeptide sites that could be phosphorylated by CDK1 (Ser⁷⁴⁹, Ser⁸⁸⁷, Ser⁹⁰⁹, Ser⁹¹⁴, Ser¹⁰⁵⁴, Ser¹⁰⁵⁹, Ser¹¹²³, Ser¹²³⁷, Ser¹²⁷⁰, and Ser¹³²¹). Alanine replacement mutations are indicated by letters. B, recombinant CUX1(612–1328/HA) proteins carrying alanine replacement mutations were expressed by transient transfections in U2OS cells and analyzed by immunoblotting. The letters above each lane indicate the alanine replacement mutations within the protein: WT (wild type), A (749), B (887), C¹ (909), C (909, 914), D (1054, 1059), F (1237, 1270), and G (1321). C–E, populations of U2OS cells stably expressing recombinant CUX1(612–1328/HA) proteins were synchronized in G₂ or M phase as described in Fig. 2A and analyzed by immunoblotting and EMSA. Where indicated, protein extracts were first incubated in the presence of λ-phosphatase (λ-ppase). The letters above each lane indicate the alanine replacement mutations within the protein, as described in A: WT (wild type), All (all SP dipeptides, CKII, PKC, PKA: 749, 887, 909, 914, 977, 992,1054,1059,1123, 1160,1175, S1215,1216, 1237,1270,1321), ABCDEFG (all SP dipeptides: 749, 887, 909, 914, 1054, 1059, 1123, 1237, 1270, 1321), FQ (1215,1216,1237,1270), PR (CKII+PKC: 977, 992, 1160, 1175), Q (PKA, 1215, 1216).