Volume 14, Issue 3, Pages 319-330 (May 2004) Mitotic Phosphorylation of the Peripheral Golgi Protein Nir2 by Cdk1 Provides a Docking Mechanism for Plk1 and Affects Cytokinesis Completion  Vladimir Litvak, Rachel Argov, Nili Dahan, Sreekumar Ramachandran, Roy Amarilio, Alla Shainskaya, Sima Lev  Molecular Cell  Volume 14, Issue 3, Pages 319-330 (May 2004) DOI: 10.1016/S1097-2765(04)00214-X

Figure 1 Nir2 Is Phosphorylated during Mitosis HeLa cells were synchronized at the G1/S boundary by double-thymidine block, washed with phosphate-buffered saline (PBS), and incubated with complete media for the indicated time periods. At each time point, half of the cells was lysed and analyzed for Nir2 expression by immunoprecipitation followed by Western blotting (A), whereas the second half was fixed in methanol and used for flow cytometric analysis (B). The propagation of the cells throughout the cell cycle is shown in (B). 2N and 4N represent DNA content. (C) Phosphatase treatment abolished the mobility shift of mitotic Nir2. Mitotic HeLa cells were prepared by nocodazole arrest along with mitotic shake-off. Nir2 was immunoprecipitated from interphase or mitotic cells and then treated with or without λ-phosphatase in the absence or presence of phosphatase inhibitors, separated by SDS-PAGE, and analyzed by Western blotting. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)

Figure 2 Cdk1 Induces a Mobility Shift of Nir2 and Phosphorylates It In Vitro (A) HeLa cells were transiently transfected with expression vectors encoding wild-type Cdk1 or its kinase-deficient mutant (Cdk1-kd) as indicated. The cells were then treated with nocodazole for 16 hr, and where indicated, olomoucine (50 μM) was added for the last 5 hr in the presence of nocodazole. Nir2 was immunoprecipitated from mitotic or interphase cells and analyzed by Western blotting using anti-Nir2 antibody. (B) Nir2-HA was transiently expressed in HEK293 either alone or with expression vectors encoding the indicated protein kinases. The mobility of Nir2 was determined by Western blotting with anti-HA antibody. (C) In vitro phosphorylation of Nir2 by Cdk1. Cdk1-HA was immunoprecipitated from mitotic or interphase HeLa cells. The immunoprecipitates were extensively washed and incubated with the indicated recombinant proteins in the presence of [γ-32P]ATP for 30 min at 30°C. The kinase reactions were separated by 12% SDS-PAGE and then subjected to autoradiography. Histone H1 (1 μg) was used as a positive control. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)

Figure 3 Nir2 Undergoes Phosphorylation on Thr-Pro Motifs during Mitosis (A) Endogenous Nir2 is phosphorylated on Thr-Pro motifs during mitosis. Endogenous Nir2 was immunoprecipitated from interphase or mitotic HeLa cells, separated by SDS-PAGE, and analyzed by Western blotting with either anti-Nir2 or anti-pTP antibodies. (B) Phosphorylation of wild-type Nir2 and its Thr/Ala mutants in interphase (I) and mitotic (M) HeLa cells was determined by immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-pThr/Pro antibody. (C) T287 is the major Thr phosphorylation site in mitotic cells. HeLa cells were transiently transfected with the indicated Nir2 mutants. The Nir2 proteins were immunoprecipitated and immunoblotted as described in (B). As shown, the phosphorylation of the double mutants T287/389A and T287/794A was significantly decreased and was hardly detected in the triple T287/389/794A mutant. (D) T287 is phosphorylated by Cdk1 in vivo. HEK293 cells were transiently transfected with the indicated Thr/Ala mutants, either alone or together with an expression vector encoding a constitutively active Cdk1 or ERK2 (ERK2-MEK1). Nir2-HA and its mutants were immunoprecipitated by anti-HA antibody, and their phosphorylation was determined by immunoblotting with anti-pTP antibody. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)

Figure 4 S382 Is the Major Phosphorylation Site of Nir2 in Mitotic Cells (A) Endogenous Nir2 is recognized by anti-MPM-2 antibody in mitotic cells. Nir2 was immunoprecipitated from interphase (I) and mitotic (M) HeLa cells, separated by SDS-PAGE, and immunoblotted with anti-Nir2 or anti-MPM-2 antibodies. (B) Mitotic phosphorylation of Nir2 mutants. HeLa cells were transiently transfected with expression vectors encoding wild-type Nir2-HA or the indicated mutants. The Nir2 proteins were immunoprecipitated from interphase or mitotic cells, and their phosphorylation on Ser/Thr-Pro motifs was determined by immunoblotting with anti-MPM-2 antibody. (C) Substitution of S382 by alanine reduces, but does not abolish, the mitotic phosphorylation of Nir2. HeLa cells were transiently transfected with expression vectors encoding the wild-type Nir2-HA or the indicated Nir2 mutants. The Nir2 proteins were immunoprecipitated from interphase or mitotic cells, and their phosphorylation on Ser/Thr-Pro motifs was determined by immunoblotting with anti-MPM-2 antibody. As shown, substitution of S382 by alanine reduced the mitotic phosphorylation but did not abolish it. The double mutant T287/S382A showed weaker phosphorylation than the S382A mutant yet exhibited stronger phosphorylation than the 5NA mutant. Phosphorylation of the triple mutant T287/S382/S896A was hardly detected, and the 10A mutant was undetectably phosphorylated. (D) S382 is phosphorylated by Cdk1 in vivo. HEK293 cells were transiently transfected with expression vectors encoding wild-type Nir2-HA or the indicated mutants, either alone or with a constitutively active Cdk1. The Nir2 proteins were immunoprecipitated by anti-HA antibody, and their phosphorylation was determined by immunoblotting with anti-MPM-2 antibody. (E) Cdk1 phosphorylates S382 in vitro. Wild-type (wt) or a kinase-deficient (kd) mutant of Cdk1-HA was transiently expressed in HEK293 cells, immunoprecipitated by anti-HA antibody, and incubated with the indicated recombinant proteins in the presence of [γ-32P]ATP for 30 min at 30°C. The kinase reactions were separated by 10% SDS-PAGE and then subjected to autoradiography. The 4NA mutant consists of the following mutations: T287/S300/S326/T389A. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)

Figure 5 Subcellular Localization of Phospho-Nir2 at S382 during Cell Division (A) Specificity of the anti-pS382 antibody. Wild-type Nir2-HA or the S382A mutant was transiently expressed in HEK293 cells, either alone or with a constitutively active Cdk1. The Nir2 proteins were immunoprecipitated and immunoblotted as indicated. (B) Localization of Nir2 and phospho-Nir2 in HeLa cells. HeLa cells, which express the Golgi protein NAGT-I fused to GFP, were immunostained with either anti-Nir2 (against aa 287–300) or anti-pS382 antibodies and analyzed by confocal microscopy. The Nir2 protein is shown in red and colocalization appears in yellow. Bar, 10 μm. (C) Phosphorylation of Nir2 by Cdk1 facilitates its dissociation from Golgi membranes. Isolated Golgi membranes (10 μg) were incubated with increasing concentrations of purified GST-Nir2, GST, or GST-Nir2 that had been phosphorylated by Cdk1 in vitro. The association of the recombinant proteins with the Golgi membranes (pellet) was determined by immunoblotting with anti-GST antibody. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)

Figure 6 Nir2-Plk1, but Not Nir2-RhoA, Interaction Is Dependent upon Nir2 Mitotic Phosphorylation (A) Mitotic Nir2 binds PBD in vitro. Lysates from nocodazole- or thymidine-arrested HeLa cells that express the wild-type Nir2-HA protein, the S382A, or the 10A mutant were incubated with a recombinant GST-fusion protein encoding the PBD (aa 326–603) of Plk1 immobilized on glutathione-agarose beads. The binding of Nir2 proteins to PBD was determined by immunoblotting with anti-HA antibody. (B) Coimmunoprecipitation of Nir2 and Plk1 in mammalian cells. Wild-type Nir2-HA, or the S382A or 10A mutants were coexpressed with Myc-tagged Plk1 in HeLa cells. The association of the Nir2 proteins with Plk1, in thymidine-arrested (T) or mitotic (M) cells, was determined by immunoprecipitation with anti-Myc or anti-HA and immunoblotting with anti-HA or anti-Myc, respectively. (C) Coimmunoprecipitation of endogenous Nir2 and Plk1 in mitotic cells. Nir2 or Plk1 was immunoprecipitated from thymidine- or nocodazole-arrested HeLa cells, and the presence of Nir2 in Plk1 immunocomplex was determined by immunoblotting with anti-Nir2 antibody, while the presence of Plk1 in Nir2 immunocomplex was determined by immunoblotting with anti-Plk1 antibody. Preimmune serum (P.I.) was used as a control for the Nir2-immune serum (I) in the immunoprecipitation assay. (D) Direct binding of mitotic Nir2 to PBD. Wild-type Nir2 or the indicated mutants were immunoprecipitated from thymidine (T)- or nocodazole (N)-arrested HeLa cells. The proteins were transferred to nitrocellulose membrane and probed with a recombinant GST-PBD fusion protein, followed by anti-GST antibody (upper panel). The same membrane was stripped and reprobed with anti-HA antibody to assess the expression levels of the Nir2 proteins (lower panel). (E) Plk1 phosphorylates Nir2 fragment (aa 205–424) in vitro. Myc-tagged wild-type Plk1 or its kinase-deficient (kd) mutant was expressed in HEK293 cells, immunoprecipitated by anti-Myc antibody, and then incubated with the indicated recombinant proteins in the presence of [γ-32P]ATP for 30 min at 30°C. The kinase reactions were separated by 10% SDS-PAGE and then subjected to autoradiography. (F) The 10A mutant normally interacts with RhoA in mitotic cells as determined by coimmunoprecipitation (left panel) and colocalizes with RhoA in the midbody (right panel). Bar, 10 μm. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)

Figure 7 Ectopic Expression of the 10A Mutant Affects Cytokinesis (A) The Nir2 10A mutant induces multinucleate cell formation. HeLa cells were transiently transfected with DNA encoding HA-tagged wild-type Nir2 or the 10A mutant. The cells were fixed 2 days later and immunostained with anti-HA antibody. Nuclei were stained with Hoechst 33258 and visualized by fluorescence microscopy. The percentage of cells bearing multinuclei was scored (left panel) for wild-type Nir2 transfectants (n = 665), the 10A mutant (n = 703), and the control nontransfected cells (n = 1541). Cells expressing the 10A mutant are shown in the right panel, along with their nuclear content. Bar, 10 μm. (B) The effects of the 10A mutant on telophase cells. The percentage of aberrant telophase cells, characterized by incomplete chromosome pair separation or unusual nuclei, was scored (left panel) for wild-type Nir2 transfectants (n = 71), the 10A mutant (n = 73), and the control nontransfected cells (n = 78). Representative images of 10A-expressing cells are shown in the right panel. Bar, 10 μm. (C) Effect of the 10A mutant on furrow ingression. Shown are representative images of Plk1 localization in the control or 10Aexpressing HeLa cells along with their nuclei. As shown, no ingression of the cleavage furrow is evident in the 10A-expressing cells. Bar, 10 μm. (D) Localization of Nir2 and Plk1 during cytokinesis. Shown are confocal images of HeLa cells double-immunostained with anti-Nir2 antibody (red) and anti-Plk1 antibody (green). Colocalization appears in yellow. Bar, 10 μm. Molecular Cell 2004 14, 319-330DOI: (10.1016/S1097-2765(04)00214-X)