Integrative Transcriptome and Proteome Study to Identify the Signaling Network Regulated by POPX2 Phosphatase

POPX2 is a serine/threonine phosphatase belonging to the protein phosphatase 2C (PP2C) family that has been found to be elevated in invasive breast cancer cells. Silencing of POPX2 results in lower cell motility and invasiveness. The molecular mechanism of POPX2-regulated cell motility is not well understood. To identify the relevant signaling pathways, we investigated the transcriptome and proteome of POPX2-knockdown MDA-MB-231 breast cancer cells. Our data suggest that POPX2 might be involved in the regulation of focal adhesions and cytoskeleton dynamics through the regulation of MAP kinase (MAPK1/3) and glycogen synthase kinase 3 (GSK3α/β) activities. Silencing POPX2 alters phosphorylation levels of MAPK1/3 and GSK3α/β and results in reduced activity of these kinases. Both MAPK and GSK3 are known to regulate the activities of transcription factors. MAPK1/3 are also implicated in the phosphorylation of stathmin. The level of phospho-stathmin was found to be lower in POPX2 knockdown cells. As phosphorylation of stathmin inhibits its microtubule severing activity, we observed less stable microtubules in POPX2 knockdown cells. Taken together, our data suggest that POPX2 might regulate cell motility through its regulation of the MAPK1/3, leading to changes in the cytoskeleton and cell motility

Integrative Transcriptome and Proteome Study to Identify the Signaling Network Regulated by POPX2 Phosphatase

POPX2 is a serine/threonine phosphatase belonging to the protein phosphatase 2C (PP2C) family that has been found to be elevated in invasive breast cancer cells. Silencing of POPX2 results in lower cell motility and invasiveness. The molecular mechanism of POPX2-regulated cell motility is not well understood. To identify the relevant signaling pathways, we investigated the transcriptome and proteome of POPX2-knockdown MDA-MB-231 breast cancer cells. Our data suggest that POPX2 might be involved in the regulation of focal adhesions and cytoskeleton dynamics through the regulation of MAP kinase (MAPK1/3) and glycogen synthase kinase 3 (GSK3α/β) activities. Silencing POPX2 alters phosphorylation levels of MAPK1/3 and GSK3α/β and results in reduced activity of these kinases. Both MAPK and GSK3 are known to regulate the activities of transcription factors. MAPK1/3 are also implicated in the phosphorylation of stathmin. The level of phospho-stathmin was found to be lower in POPX2 knockdown cells. As phosphorylation of stathmin inhibits its microtubule severing activity, we observed less stable microtubules in POPX2 knockdown cells. Taken together, our data suggest that POPX2 might regulate cell motility through its regulation of the MAPK1/3, leading to changes in the cytoskeleton and cell motility

Supplementary Materials from LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains

Supplementary Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 1

Phosphatase POPX2 Exhibits Dual Regulatory Functions in Cancer Metastasis

Cancer metastasis is a complex mechanism involving multiple processes. Previously, our integrative proteome, transcriptome, and phosphoproteome study reported that the levels of serine/threonine phosphatase POPX2 were positively correlated with cancer cell motility through modulating MAPK signaling. Surprisingly, here we found that POPX2 knockdown cells induced more numerous and larger tumor nodules in lungs in longer term animal studies. Interestingly, our analysis of DNA microarray data from cancer patient samples that are available in public databases shows that low POPX2 expression is linked to distant metastasis and poor survival rate. These observations suggest that lower levels of POPX2 may favor tumor progression in later stages of metastasis. We hypothesize that POPX2 may do so by modulation of angiogenesis. Secretome analysis of POPX2-knockdown MDA-MB-231 cells using LC–MS/MS-based SILAC quantitative proteomics and cytokine array show that silencing of POPX2 leads to increased secretion of exosomes, which may, in turn, induce multiple pro-angiogenic cytokines. This study, combined with our previous findings, suggests that a single ubiquitously expressed phosphatase POPX2 influences cancer metastasis via modulating multiple biological processes including MAPK signaling and exosome cytokine secretion

Aberrant Aurora B kinase activation upon RanGTP depletion leads to aberrant chromosomal alignment.

<p>A) Mitotic tsBN2 cells incubated at permissive or non-permissive temperature were analyzed by immunofluorescence staining with anti-Aurora B kinase and anti-Aurora B kinase (pThr232). Scale bar: 10 µm. B) Magnified images of the boxed regions illustrating anti-Aurora B kinase and anti-Aurora B kinase (pThr232) staining. Magnified merged image is exclusive of DNA. C) Western blot analysis of mitotic tsBN2 cells incubated at permissive or non-permissive temperature and harvested via mechanical shake-off. Actin was used as loading control. D) Quantified Aurora B kinase (pThr232) intensities were normalized and presented as relative fold change ± s.d. (error bar) of 3 independent experiments. E) Aurora B kinase assay was conducted using Aurora B kinase protein immunoprecipitated from mitotic tsBN2 cells incubated at permissive or non-permissive temperature. Kinase activity was determined by phosphorylation of a known Aurora B kinase substrate, Histone H3. F) Quantified histone H3 (pSer10) intensities were normalized and presented as relative fold change ± s.d. (error bar) of 3 independent experiments. G) Time-lapse imaging of metaphase tsBN2 cells expressing H2B-GFP and tubulin-mCherry. Control experiment (upper panel), temperature-shift experiment (middle panel), and temperature-shift + ZM447439 (bottom panel). Scale bar: 10 µm.</p

Crm1-Mst1-Aurora B kinase axis dictates the maintenance of stable kinetochore-microtubule attachments.

<p>A) Mitotic tsBN2 cells were immunostained with anti-Crm1 and anti-Mst1 following incubation at permissive or non-permissive temperature. B) Magnified images of the boxed regions illustrating anti-Crm1 and anti-Mst1 staining (magnified merged image is exclusive of DNA). C) Quantified Crm1 and Mst1 intensities were normalized and presented as relative fold change ± s.d. (error bar) of three independent experiments. D) Co-immunoprecipitation assay was conducted using monoclonal anti-Mst1 antibody on mitotic tsBN2 cell lysates harvested 4 hours after incubation at permissive or non-permissive temperature. E) Quantified Crm1 intensities were normalized and presented as relative fold change ± s.d. (error bar) of three independent experiments. F) Time-lapse imaging of metaphase tsBN2 cells expressing H2B-GFP and Mst1 WT-mCherry, Mst1 K59R-mCherry or mCherry (positive control). Overexpression of Mst1 WT abrogated the misalignment phenotype in cells incubated at non-permissive temperature. Arrows pointed to misaligned chromosomes. Scale bar: 10 µm.</p

Mitotic RanGTP is required for proper kinetochore-microtubule attachments during metaphase.

<p>A) Metaphase tsBN2 Cells were immunostained with anti-centromeric antigens (ACA) and tubulin antibodies. Magnified images show end-on kinetochore-microtubule attachments (control, upper panel) and unattached chromosomes (temperature-shifted, lower panel). B) Histogram shows total cold-stable microtubule intensity (A.U.) after cold-induced microtubule depolymerization. Error bars show ± s.d. (Student’s t-test). C) Representative images of cold-treated mitotic spindles from mitotic tsBN2 cells incubated at permissive or non-permissive temperature. Immunostaining of spindle checkpoint protein BubRI with D) tubulin or E) anti-centromeric ACA. Scale bar: 10 µm.</p

Robust interaction of functionally active Mst1 with Aurora B kinase for its inhibitory effect.

<p>A) Co-immunoprecipitation assay conducted using anti-Mst1 antibody on mitotic tsBN2 cell lysates harvested 4 hours after incubation at permissive or non-permissive temperature. B) Quantified Aurora B kinase intensities were normalized and presented as relative fold change ± s.d. (error bar) of three independent experiments. C) Immunoprecipitation assay conducted using monoclonal anti-FLAG antibody on metaphase-enriched HEK cell lysates co-transfected with plasmids as indicated. D) Quantified Aurora B kinase intensities were normalized against immunoprecipitated Aurora B kinase co-transfected with FLAG-Mst1 WT (middle lane) and presented as relative fold change ± s.d. (error bar) of three independent experiments. E) Immunoprecipitation assay conducted using monoclonal anti-FLAG antibody on metaphase-enriched HEK cell lysates co-transfected with plasmids as indicated. F) Quantified Aurora B kinase intensities were normalized against immunoprecipitated Aurora B kinase co-transfected with FLAG-Mst1 WT (middle lane) and presented as relative fold change ± s.d. (error bar) of three independent experiments. Asterisk (*) indicates non-specific bands. G) Metaphase spread of tsBN2 cells expressing Mst1 WT or K59R ΔC-mCherry were immunostained with anti-Aurora B kinase. Scale bar: 2 µm. Images were acquired with fixed-exposure mode. H) Histogram shows percentage of metaphase chromosomes with Mst1-mCherry fusion protein. Error bars show ± s.d. from three independent experiments. I) Western blot analysis of metaphase-enriched HEK cells co-transfected with Aurora B kinase and FLAG-Mst1 as indicated. Asterisk (**) indicates endogenous Mst1. Actin was used as loading control. J) Quantified Aurora B kinase (pThr232) intensities were normalized and presented as relative fold change ± s.d. (error bar) of three independent experiments.</p

Model illustrating the role of mitotic RanGTP in sustaining stable chromosomal alignment during metaphase.

<p>In the presence of high mitotic RanGTP, Crm1 is localized to the kinetochores. Mst1 is then recruited as part of the RanGTP-Crm1-Mst1 ternary complex. The presence of Mst1 limits the autophosphorylation of Aurora B kinase and thus stabilizes kinetochore-microtubule attachments, which, results in stable chromosome alignment at the metaphase plate (A). When RanGTP is depleted, Crm1 is unable to bind and target Mst1 to the kinetochore. Subsequently, Aurora B kinase becomes hyperactivated to promote kinetochore-microtubule reorientation. The loss of proper amphitelic attachment ensues leading to the displacement of metaphase chromosomes from the equator (B).</p

RanGTP is depleted at non-permissive temperature and affects maintenance of chromosome alignment in tsBN2 cells.

<p>A) Schematic depiction of the experimental conditions. Cells were arrested at metaphase using MG132 for 2 hours, prior to incubation at permissive (33.5°C) or non-permissive temperature. B) tsBN2 cells expressing Rango and H2B-mCherry. C) tsBN2 cells expressing Rango and tubulin-mCherry. Control experiments at 33.5°C and temperature-shift experiments at 39.5°C. Color bar represents FRET intensity. Scale bar: 10 µm. D) Line chart representation the Rango FRET ratio (according to Youvan’s method) at various time-lapse intervals for control, 33.5°C (round markers), and temperature-shifted, 39.5°C (square markers) cells. Error bars represent ± standard deviation (s.d.). E) Western blot analysis of Rango and Ran from mitotic tsBN2 cells incubated at permissive or non-permissive temperature for various time points. Actin was used as loading control.</p