The SIN kinase Sid2 regulates cytoplasmic retention of the S. pombe Cdc14-like phosphatase Clp1

Abstract

Cdc14-family phosphatases play a conserved role in promoting mitotic exit and cytokinesis by dephosphorylating substrates of cyclin-dependent kinase (Cdk). Cdc14-family phosphatases have been best studied in yeast (for review, see [1, 2]), where budding yeast Cdc14 and its fission yeast homolog Clp1 are regulated partly by their localization; both proteins are thought to be sequestered in the nucleolus in interphase. Cdc14 and Clp1 are released from the nucleolus in mitosis, and in late mitosis conserved signaling pathways termed the mitotic exit network (MEN) and the septation initiation network (SIN) keeps Cdc14 and Clp1, respectively, out of the nucleolus through an unknown mechanism [3-6]. Here we show that the most downstream SIN component, the Ndr-family kinase Sid2, maintains Clp1 in the cytoplasm in late mitosis by phosphorylating Clp1 directly and thereby creating binding sites for the 14-3-3 protein Rad24. Mutation of the Sid2 phosphorylation sites on Clp1 disrupts the Clp1-Rad24 interaction and causes Clp1 to return prematurely to the nucleolus during cytokinesis. Loss of Clp1 from the cytoplasm in telophase renders cells sensitive to perturbation of the actomyosin ring but does not affect other Clp1 functions. Because all components of this pathway are conserved, this might be a broadly conserved mechanism for regulation of Cdc14-family phosphatases.

Figure 1. Sid2 phosphorylation of Clp1 promotes binding of Rad24 (14-3-3) to Clp1 in vitro

(A) In vitro kinase assays [23] were performed by using Sid2 kinase complexes from TAP (tandem-affinity purification) eluates from S. pombe cells, and bacterially expressed MBP-Clp1. Protein labeled by γ-³²P was detected using a Phospho Imager (Molecular Dynamics), and the gel was stained with Coomassie Blue (CB) as loading control. (B) MBP-Clp1 was pre-incubated with Sid2 kinase in the presence or absence of unlabeled ATP, and then incubated with bacterial lysates expressing GST or GST-Rad24. Glutathione sepharose resin was added, and the precipitates were detected by Western blot using anti-MBP antiserum (New England BioLabs). (C) Phosphoamino acid analysis of MBP-Clp1 phosphorylated by Sid2 kinase. The positions of the phospho-threonine and phospho-tyrosine standards are indicated by circles. (D) Phospho-tryptic peptide analysis of MBP-Clp1 and MBP-Clp1-5A phosphorylated by Sid2 kinase. The positions of six major phosphopeptides are numbered. The position of the origin was indicated with an “x”. The anode is on the left. (E) In vitro phosphorylation sites of Clp1 by Sid2 kinase identified by mass spectrometry are listed. (F) MBP-Clp1, MBP-Clp1-5A, MBP-Clp1-6A, and MBP-Clp1-7A were purified from bacterial lysates, and phosphorylated with Sid2 kinase purified using anti-Myc antibody from cdc16-116 sid2-13Myc cells. (G) Phosphatase activity of MBP-Clp1, MBP-Clp1-C286S (phosphatase inactive allele), MBP-Clp1-6A, and MBP-Clp1-7A were determined by their ability to hydrolyze DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) [24]. Reactions were performed in triplicate for standard error analysis. Data are representative of two independent experiments. (H) Cell lysates of clp1-GFP and clp1-6A-GFP were prepared in NP-40 buffer (supplemental methods). The Clp1-GFP and tubulin protein levels were determined by Western blot using anti-GFP (Santa Cruz Biotechnology), and anti-TAT1 antibodies.

Figure 2. The clp1-6A mutation disrupts SIN regulation of Clp1 nucleolar localization

(A) Time-lapse images of clp1-GFP and clp1-6A-GFP cells both expressing Rlc1-GFP as an actomyosin ring marker were collected every 5 minutes, using a spinning disc confocal microscope. Ten stacks of images were captured for each time point, with a step size of 0.55 μm between focal planes. Nucleolar to cytoplasmic ratios were calculated and shown in Figure S2C. (B) clp1-GFP and clp1-6A-GFP cells were grown to mid-log phase and treated with 4μM Latrunculin B (Sigma). Cells were collected every 30 minutes, and subjected to methanol fixation and DAPI staining (shown in red). Localization of Clp1-GFP and Clp1-6A-GFP are shown after 180 min (left panel). Cells with nucleolar or dispersed GFP localization were quantified over time (right panel). (C) clp1-GFP and clp1-6A-GFP in a cdc16-116 temperature sensitive background were cultured to mid-log phase at 25°C, then shifted to 36°C for 2 hr. The cells were subjected to methanol fixation and DAPI staining (shown in red). Quantification of nucleolar or dispersed localization of Clp1-GFP and Clp1-6A-GFP was scored in binucleate septated cdc16-116 cells. (D) Protein lysates prepared from clp1-GFP and clp1-6A-GFP cells grown at 30°C were split 3 ways. Clp1 was immunoprecipitated from one sample (IP) using a mouse monoclonal anti-GFP antibody (Molecular Probes), and the other 2 samples were mixed with bacterially produced GST (GST), or GST-Rad24 (GST-Rad24) (supplemental methods). The complexes were precipitated with glutathione sepharose resin and probed, along with the immunoprecipitated sample, by Western blot using anti-GFP antibodies.

Figure 3. Functional analysis of Clp1-6A in cytokinesis

(A) clp1-6A-GFP in different actomyosin ring mutant backgrounds (cdc15-140, myo2-E1, and mid1-18) were grown to mid-log phase, spotted on YE plates in 10-fold serial dilutions, and incubated at 25°C, 30°C, 33°C, and 36°C as indicated. (B) clp1-GFP, clp1Δ, clp1-6A-GFP, and clp1-C286S-13Myc were grown to mid-log phase, and spotted in 10-fold serial dilutions on YE plates containing 3μM LatB or DMSO (solvent control). The plates were incubated at 30°C for 3 days.

Figure 4. A model of Clp1 regulation by Sid2 kinase and Rad24