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. 2013 Sep 1;12(17):2876-87.
doi: 10.4161/cc.25919. Epub 2013 Aug 2.

Protein Phosphatase 4 Is Phosphorylated and Inactivated by Cdk in Response to Spindle Toxins and Interacts With γ-Tubulin

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Free PMC article

Protein Phosphatase 4 Is Phosphorylated and Inactivated by Cdk in Response to Spindle Toxins and Interacts With γ-Tubulin

Martin Voss et al. Cell Cycle. .
Free PMC article

Abstract

Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G 1/S to the G 2/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.

Keywords: Cdk1; cell cycle; centrosome; nocodazole; paclitaxel; protein phosphatase 4; γ-tubulin.

Figures

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Figure 1. Phosphorylation of the regulatory subunits of Ppp4c in response to spindle toxins and a cell cycle inhibitor. (A) Human FLAG-R2 was expressed in HEK293 cells cultured with or without nocodazole treatment for 16 h. Cells were lysed in lysis buffer containing phosphatase inhibitor. FLAG-tagged R2 was immunoadsorbed from the cell lysates and the proteins in the immunopellets separated by gel electrophoresis. The R2 protein band was cut out of the gel, digested with trypsin and the R2 peptides were analyzed for phosphorylation by precursor ion scanning. The spectra show two phosphorylated R2 peptides that are present in nocodazole treated samples and virtually absent in untreated controls, while a third R2 peptide and the FLAG peptide are present at similar levels in untreated and nocodazole treated samples. The ion at m/z 448.2 was singly charged and did not correspond to the mass of any expected peptide from R2. The sequences of the R2 peptides are presented on the right. The sequence of a R3A phosphorylated peptide, which was also identified after treatment of cells with nocodazole, is shown below. m/z, mass/charge; amu, atomic mass units; cps, counts per second. (B and C) Phosphorylation of endogenous R2 and R3A in response to cell cycle inhibitors in HEK293 and HeLa cells, untreated (control) or treated with 300 nM nocodazole, 25 μM roscovitine or 33 nM paclitaxel for 16 h. Proteins in the cell lysates were separated by SDS gel electrophoresis, transferred to nitrocellulose membranes and probed with antibodies to the indicated proteins. Size markers in kDa are shown on the left. The predicted molecular sizes are human R3A 95.4 kDa, R2 46.9 kDa and Ppp4c 35.1 kDa (Q6IN85, Q9N27, P60510 at http://www.uniprot.org/).
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Figure 2. Effect of cell cycle inhibitors on the subcellular location of R2 and R3A. HeLa cells were fixed and initially viewed using differential interference contrast (DIC). DNA (blue) was stained with DAPI. Anti-pericentrin stain (green) identifies the pericentriolar material. Anti-Ppp4c(287–305) stain (red) in the top panel and anti-R3A(819–833) stain (red) in the middle panel identifies Ppp4c and R3A respectively as indicated. The top panel shows Ppp4c enrichment in the pericentriolar region in control and roscovitine treated cells in metaphase and cells treated with nocodazole and paclitaxel. The middle panel shows R3A enrichment in the pericentriolar region (control) and the loss or partial loss of R3A from the pericentriolar region after treatment of the cells with roscovitine. Representative images are shown in each case. The bottom, panel shows the control images, where anti-Ppp4c or anti-R3A antibodies are blocked with their respective peptide immunogen. Scale bars are 10 μM.
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Figure 3. Effect of kinase inhibitors on the nocodazole-induced phosphorylation of endogenous R2 and R3A in HEK293 cells. (A–D) HEK293 cells at 60–80% confluence were cultured at 37 °C untreated (con) or treated with 300 nM nocodazole (Noc) for 16 h. The following protein kinase inhibitors were added to the culture medium to the stated concentrations one hour prior to the end of the nocodazole treatment: 10 μM BIRB 0796 (BIRB); 2 μM PD 0325901 (PD); 1 μM PI-103; 2 μM GDC-0941 (GDC); 10 μM Harmine (Har); 25 μM roscovitine (Ros), except in the right hand lane (Ros +Noc) of (D) where roscovitine was present one hour prior to the start and during 16 h nocodazole treatment. Proteins in the cell lysates were separated by SDS gel electrophoresis, transferred to nitrocellulose membranes and probed with antibodies to the indicated proteins.
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Figure 4. Phosphorylations of endogenous R2 and R3A during progression through the HeLa cell cycle. (A) Analysis of HeLa cells by flow cytometry shows the distribution of cells in asynchronous cultures, the same cultures immediately following the double thymidine-block, 10 h after release from the double thymidine-block and cell cultures blocked at G2/M with nocodazole. (B) Immunoblots showing endogenous R2pSer159 in R2 immunopellets. M indicates the marker proteins, with their size in kDa. (C) R2pThr173 in lysates from asynchronous cultures (AS), immediately following the double thymidine-block (Thy), 10 h after release from the double thymidine-block (10 h release) and in cells treated with nocodazole (Noc). (D) Immunoblot analysis of the endogenous R2, R2pThr173 and endogenous R3A during the cell cycle after a double thymidine-block. Cyclin E detection indicates that the cells were blocked at G1/S by thymidine and histone H3 Ser10 phosphorylation that cells were at G2/M. GAPDH was used as a control for sample loading.
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Figure 5. Phosphorylation of Ppp4 regulatory subunits and assay of endogenous Ppp4 activity during the HEK293 cell cycle. (A) Immunoblots showing endogenous R2pSer159 and R3pSer741 in R3A immunopellets from asynchronous cultures (AS), in cells following release from a double thymidine-block and in cells treated with nocodazole (Noc) or paclitaxel (Tax). Control samples were prepared similarly except that the anti-R3A antibody was incubated with the peptide immunogen for 30 min prior to use. (B) Immunoblots showing endogenous R2pSer159 and R3pSer741 in R2 immunopellets from asynchronous cultures, in cells following release from a double thymidine-block and in cells treated with nocodazole or paclitaxel. Control samples were prepared similarly except that the anti-R2 antibody was incubated with the peptide immunogen for 30 min prior to use. (C) Protein phosphatase activity was assayed in anti-R3A immunopellets from lysates of the HEK293 cells at 0, 6, 10, and 16 h after release from a double thymidine-block. The phosphatase activity in the immunopellets, determined using the phosphopeptide RRApTVA as substrate. The graph shows the mean values and error bars indicate ± SD of 4 assays for each time point. (D) Affinity purified recombinant GST-R3A-His6R2-Ppp4c was treated with λ phosphatase (1 μg/ml) and subsequently with Cdk1-cyclin B (1 μg/ml) at 30 °C for 30 min. and analyzed by SDS-PAGE and immunoblotting. (E) Recombinant GST-R3A-His6R2-Ppp4c treated with Cdk1-cyclin B shows the phosphorylation of R2 Ser159 and R2Thr173
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Figure 6. Identification of Ppp4c interacting proteins. (A) Immunoblots showing endogenous γ-tubulin and TUBGCP2 but not TUBGCP3 in immunopellets of endogenous Ppp4c complexes from lysates of asynchronous HEK293 cells (AS) or cells treated with nocodazole or paclitaxel. The control samples were prepared similarly except that the anti-Ppp4c antibody was incubated with the peptide immunogen for 30 min prior to use. (B) Endogenous Ppp4c complexes were immunoadsorbed using anti-Ppp4c antibody-Sepharose beads from HEK293 lysates (containing 100 nM microcystin) after release from a double thymidine cell cycle block. Proteins in the washed immunopellets were examined by SDS-PAGE. (C) Immunoblots showing endogenous γ-tubulin and TUBGCP2 in immunopellets of endogenous Ppp4c complexes from lysates of asynchronous HEK293 cells after three washes in lysis buffer [wash 1], three washes in lysis buffer, followed by one wash in no salt buffer (50 mM TRIS-HCl pH 7.5, 0.1 mM DTT, 0.1 mM EGTA) and one wash in high salt buffer (50 mM TRIS-HCl pH 7.5, 0.1 mM DTT, 500 mM NaCl) [wash 2] and three washes in lysis buffer, followed by two washes in no salt buffer and two washes in high salt buffer [wash 3]. (D and E) γ-tubulin was phosphorylated in vitro by Cdk1-cyclin B or Brsk1 and then dephosphorylated by Ppp4c-R2-R3A. The γ-tubulin samples were examined by SDS-PAGE and the γ-tubulin bands cut out of the gel, digested with trypsin and the peptides analyzed by mass spectrometry on an LTQ-Orbitrap Classic. (D) Extracted Ion Chromatograms (XICs) for an m/z of 699.3290 show the elution profile of a peptide identified by MSMS analysis as the phosphorylated peptide 195 L(pT)QNADCVVVLDNTALNR-212, eluting at 32.17 min (indicated by an asterisk*). The relative amounts under the different conditions were determined from the area under the relevant peak. Controls: γ-tubulin, γ-tubulin+Ppp4 complex. (E) XICs for an m/z of 711.3621 show the elution profile of a second phosphorylated peptide, 73-VIHSILN(pS)PYAK-84, again identified by MSMS, eluting at 25.15 min (indicated by an asterisk*). The relative amounts under the different conditions were determined from the area under the relevant peak. The peak eluting at 34.5 min could not be identified.
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Figure 7. Regulation of the Ppp4c-R2-R3 complex by phosphorylation. (A) Comparison of the N-terminal half of human (Hs), Mus musculus (Mm), Drosophila melanogaster (Dm), and Saccharomyces cerevisiae (Sc) R2 sequences showing Ser159 and Thr173 phosphorylation sites (indicated by arrows) in human and murine R2 and the orthologous Ser phosphorylation in Drosophila. (B) Model showing the Cdk1-cyclin B phosphorylations of R2Ser159, R2Thr173 and R3ASer741, which decrease Ppp4c-R2-R3A activity between G1/S and M-phase of the cell cycle (upper part of the figure). Ppp4c-R2-R3A interacts with γ-tubulin and TUBGCP2 (two components of γ-TuSC) keeping the protein(s) dephosphorylated in G1, but allowing the them to be phosphorylated between G1/S and M-phase and nucleate microtubule growth, (γ-tubulin may be phosphorylated on Ser80 and Thr196). When Cdk1-cyclin B activity decreases at the end of M phase, Ppp4c-R2-R3A is dephosphorylated and activated and γ-TuSC will be dephosphorylated, inhibiting microtubule nucleation. In response to the spindle toxins, nocodazole and paclitaxel, the cell cycle is blocked at M-phase with Cdk1-cyclin active, enabling the kinase to phosphorylate R2Ser159, R2Thr173, and R3ASer741 and inactivate Ppp4c-R2-R3A. γ-TuSC will undergo prolonged phosphorylation and may be ubiquitylated, because of the cell cycle block. The cell cycle then cannot proceed unless the spindle toxin is removed. Brsk1/SADB may indirectly phosphorylate γ-tubulin on Ser131.,

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