Regulation of a LATS-homolog by Ras GTPases is important for the control of cell division

Abstract

Background: Nuclear Dbf-related/large tumor suppressor (NDR/LATS) kinases have been shown recently to control pathways that regulate mitotic exit, cytokinesis, cell growth, morphological changes and apoptosis. LATS kinases are core components of the Hippo signaling cascade and important tumor suppressors controlling cell proliferation and organ size in flies and mammals, and homologs are also present in yeast and Dictyostelium discoideum. Ras proto-oncogens regulate many biological functions, including differentiation, proliferation and apoptosis. Dysfunctions of LATS kinases or Ras GTPases have been implicated in the development of a variety of cancers in humans.

Results: In this study we used the model organism Dictyostelium discoideum to analyze the functions of NdrC, a homolog of the mammalian LATS2 protein, and present a novel regulatory mechanism for this kinase. Deletion of the ndrC gene caused impaired cell division and loss of centrosome integrity. A yeast two-hybrid analysis, using activated Ras proteins as bait, revealed NdrC as an interactor and identified its Ras-binding domain. Further in vitro pull-down assays showed that NdrC binds RasG and RasB, and to a lesser extent RasC and Rap1. In cells lacking NdrC, the levels of activated RasB and RasG are up-regulated, suggesting a functional connection between RasB, RasG, and NdrC.

Conclusions: Dictyostelium discoideum NdrC is a LATS2-homologous kinase that is important for the regulation of cell division. NdrC contains a Ras-binding domain and interacts preferentially with RasB and RasG. Changed levels of both, RasB or RasG, have been shown previously to interfere with cell division. Since a defect in cell division is exhibited by NdrC-null cells, RasG-null cells, and cells overexpressing activated RasB, we propose a model for the regulation of cytokinesis by NdrC that involves the antagonistic control by RasB and RasG.

Figure 1

NdrC interacts with Ras proteins. A. In yeast two-hybrid experiments with various activated Ras proteins as bait, NdrC was revealed as a strong interactor of RasG and Rap1. Domain organization of NdrC, and mapping of the Ras binding domain (RBD, aa 107–284), NTR (N-terminal regulatory domain, aa 650–710), phosphorylation site (T703), catalytic domain (kinase domain aa 718–1019, subdomains I-X), I (insert, aa 867–913), AS (activation segment, aa 914–928; regulatory phosphorylation site S917), HM (hydrophobic motif aa 1092–1099; phosphorylation site T1095). B. Binding of GST-NdrC-RBD to Ras proteins in vivo. The RBD of NdrC was used to detect Ras proteins in Dictyostelium cell lysates as described in Methods. The bound material was analyzed by Western blotting using antibodies specific to RasB, RasG, RasC and Rap1 (upper panel). GST-only was tested in all pull-down experiments, and consistently there was no binding (here shown only for RasB). Equal sample loading was verified by staining of a duplicate gel with Coomassie Blue (lower panel); only the range of the strongest band is shown. C. Representative Western blot showing the pull-down of His-tagged Ras proteins by GST-tagged NdrC-RBD. Recombinant His-tagged Ras proteins (constitutively GTP- or GDP-bound) were allowed to bind in vitro to GST-tagged bacterially expressed NdrC-RBD. For details of the quantitative assay please see Methods. The amount of bound Ras proteins was detected by Western blotting using an anti-His Tag antibody.

Figure 2

NdrC-null cells are impaired in cell division. A. Gene replacement of ndrC (DDB0219984) by a blasticidin-S resistance cassette. P1-P2 and P1-P3 indicate the primer combinations that were used initially to identify gene knockouts by PCR. B. Live-cell microscopy of wild-type (left) and ndrC-null cells. ndrC-null cells are much larger than wild-type cells, and often divide by traction-mediated cytofission (as shown in the right image). C. Fixed wild-type cells stained with TRITC-phalloidin for actin and TO-PRO-3 for DNA. D. Fixed ndrC-null cell stained with TRITC-phalloidin for actin and TO-PRO-3 for DNA. E. Histogram showing the percentage of cells carrying the indicated numbers of nuclei in wild-type and ndrC-null mutants. Cells were grown in Petri dishes, fixed, stained, and for each strain the nuclei of >500 cells were counted. The counting was repeated in an independent experiment with almost identical results. Bars, 10 μm.

Figure 3

Cells lacking NdrC show centrosomal aberrations. A. In comparison to wild-type, B, C multinucleated ndrC-null cells frequently show supernumerous centrosomes. D. Histogram depicting the percentage of normal (centrosomes/nuclei = 1) and aberrant (centrosomes/nuclei > 1) centrosomes in wild-type and ndrC-null cells. E, F. Visualization of mitotic spindles in fixed ndrC-null cells by immunolabeling with anti-α-tubulin antibodies, and staining of DNA with TO-PRO-3. Spindle formation occurs synchronously in multinucleate cells (E), as well as in multinucleate cells with supernumerous centrosomes (F). Bars 5 μm in (A), and, 10 μm in (B), (C), (E), and (F).

Figure 4

Localization and interaction of NdrC with Ras proteins. A. NdrC localizes to the centrosome. Fixed Dictyostelium cells were immunolabeled with centrosome-specific monoclonal anti-CP224 antibodies, and polyclonal anti-NdrC antibodies. Primary antibodies were detected with anti-mouse Alexa Fluor-568 and anti-rabbit Alexa Fluor-488 antibodies. In NdrC-null cells, no staining with anti-NdrC antibodies was detected. Bar, 10 μm. B. Live-cell imaging of a Dictyostelium cell expressing GFP-NdrC-RBD. Left image, GFP-NdrC-RBD signal; right image, merge. Bar, 10 μm. C. Live-cell imaging of GFP-RasB expressing wild-type cells shows localization of GFP-RasB to the cell cortex. D. GFP-RasG localization to the cortex of wild-type cells, shown by live cell microscopy. E. GFP-RasG(G12T) localizes to the cortex and filopodia of wild-type cells. F. Cytofission of GFP-RasB(G12T) expressing wild-type cell. G. GFP-RasB(G12T) expressed in wild-type cells localizes to the cortex and filopodia (image enlargement on the right), and results in enlarged multinucleate cells. Bars in (C)-(G), 5 μm. H. GST-NdrC-RBD pull-down of RasB tagged to GFP shows interaction and thereby activity of the GFP-tagged Ras-GTPase. GFP-tagged RasG overexpressed in wild-type cells interacts with the GST-NdrC-RBD. The levels of bound RasG were detected by Western blotting using anti-GFP antibodies. I. Levels of activated Ras proteins in wild-type compared to ndrC-null cells. Total cell extracts from wild-type or ndrC-null cells were bound to GST-Byr2-RBD as described in Methods. The amount of activated Ras proteins pulled down or of total Ras protein in the lysate was determined by Western blotting using specific polyclonal antibodies against RasB, RasG, and Rap1. The data shown is for a single experiment, but similar results were obtained in two other experiments.

Figure 5

Model suggesting the regulation of NdrC by Ras GTPases as explained in the text.