ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis
Research highlights
► hDlg is phosphorylated during mitosis in multiple residues. ► Prospho-hDlg is excluded from the midbody during mitosis. ► hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. ► ERK5 pathway mediates hDlg phosphorylation in mitosis.
Introduction
Human discs-large (hDlg) protein, also called Dlg1, dlgh1 and SAP97, is an orthologue of the Drosophila tumour suppressor Dlg. It is a member of the membrane-associated guanylate kinase family of scaffold proteins, which include the post-synaptic density protein-95 (PSD95), the zonula occludens proteins (ZO1, ZO2 and ZO3) or the calcium/calmodulin dependent serine protein kinase (CASK). hDlg has multiple protein–protein interaction domains such as three PDZ domains, a SH3 domain, an L27 domain and a catalytically inactive guanylate kinase (GUK)-like region [1]. Functions of hDlg have been related to growth control, and to the establishment and maintenance of cell polarity and cell adhesion [2], [3], [4]. Moreover, gene-targeted mice lacking full-length hDlg showed defects in the morphogenesis of the kidney and urogenital tracts [5], [6].
Several studies have shown that either inactivation or depletion of the Drosophila Dlg protein results in neoplastic growth of imaginal disc epithelial cells [7]. Moreover, the expression of the Dlg mammalian counterpart (hDlg) in epithelial-derived cancers (such as cervical, gastric and colon cancers) is extremely low or even absent [8]. hDlg have been shown to interact with a number of proteins related to the cell cycle control. Thus, hDlg binds to the tumour suppressor adenomatous polyposis coli (APC) and negatively regulates cell cycle progression from G1 to S phase [9]. Also, hDlg interacts with several viral oncoproteins, such as the viral human papillomavirus (HPV) E6, the human T cell leukaemia virus type 1 (HTLV-1) Tax or the adenoviral E4 ORF1 [10]. In addition, the association of these viral proteins with hDlg regulates their oncogenic activity [10], although the mechanism by which hDlg complexes are regulated in the cell cycle is still unclear. In this regard, it has been shown that binding of HPV E6 protein to hDlg causes a decrease in hDlg protein levels by inducing its proteasome-mediated degradation [11]. In epithelial cell lines, the phosphorylation of hDlg makes it more susceptible to degradation induced by the HPV E6 [12].
Besides the indication that hDlg phosphorylation may modulate its protein levels in cells, during the last few years phosphorylation has been established as a mechanism for regulating hDlg functions and its localisation within the cell. Accordingly, numerous kinases have been shown to phosphorylate hDlg [4], [13], [14]. We have shown that in response to cell stress, hDlg is hyperphosphorylated by p38γ[15], [16]. hDlg is targeted to the cytoskeleton by its association with guanylate kinase-associated protein (GKAP), and p38γ-catalysed phosphorylation of hDlg triggers its dissociation from GKAP, releasing it from the cytoskeleton [15]. To date, PDZ-binding kinase (PBK) [17] and CDK1/2 [18] are the only kinases that regulate the cell cycle and are also linked to phosphorylation of hDlg. In addition, hDlg is phosphorylated during mitosis in HaCaT and HeLa cells but the molecular mechanisms by which hDlg is regulated during mitosis remain obscure [14], [17]. Here we addressed this question. Our data confirm that hDlg is phosphorylated in a cell cycle-dependent manner, with maximal phosphorylation at mitosis. We identified three residues phosphorylated in mitosis, some are previously unreported, and were located in the hDlg N-terminal half. Our data also show that hDlg phosphorylation in mitosis is regulated by ERK5 pathway and not by p38γ.
Section snippets
Reagents
Nocodazole was purchased from Sigma. SB203580 and SP600125 were from Calbiochem, and PD184352 and BIRB0796 were made by custom synthesis [19]. All anti-hDlg antibodies were generated as previously described [15]. Anti-JNK1/2 was from New England Biolabs; anti-ERK5 was from the Division of Signal Transduction Therapy (Dundee, UK). Anti-phospho-Ser10 Histone H3 was from Upstate and anti-Cyclin B1 from BD Pharmingen. Anti-p38γ antibody was raised and purified as described elsewhere [20]. All
hDlg is phosphorylated during mitosis
We have previously reported that, when cells are exposed to stress, endogenous hDlg is phosphorylated in the residue Ser158 by the kinase p38γ. Therefore we initiated experiments to examine hDlg phosphorylation during mitosis, using hDlg(pS158), an antibody that specifically recognises hDlg phospho-Ser158 [15]. We first analysed by microscopy hDlg phosphorylation in resting asynchronous HeLa cell and found that hDlg(pS158) stained cells that exhibit condense chromatin typical from mitotic cells
Acknowledgments
We thank Dr. R. Davis for the JNK1/2-deficient MEFs, and the protein production and antibody purification teams (Division of Signal Transduction Therapy, University of Dundee), coordinated by Dr. H. McLauchlan and J. Hastie, for antibodies. F.A.I was supported by an FPU fellowship from the Spanish Ministry of Education. The work in the author’s laboratory is supported by the Spanish Ministerio de Educación y Ciencia (MEC) Spain (BFU2007-67577).
References (31)
- et al.
Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells
J. Biol. Chem.
(2004) The maternal effect of lethal (1) discs-large-1: a recessive oncogene of Drosophila melanogaster
Dev. Biol.
(1988)- et al.
The high-risk HPV E6 oncoprotein preferentially targets phosphorylated nuclear forms of hDlg
Virology
(2009) - et al.
BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo
J. Biol. Chem.
(2005) - et al.
Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer
Mol. Cell. Proteomics
(2006) - et al.
Redistribution of the discs large tumor suppressor protein during mitosis
Exp. Cell Res.
(2003) - et al.
Functional involvement of human discs large tumor suppressor in cytokinesis
Exp. Cell Res.
(2008) - et al.
The distribution and function of alternatively spliced insertions in hDlg
J. Biol. Chem.
(2002) - et al.
Erk5 is activated and acts as a survival factor in mitosis
Cell. Signal.
(2007) - et al.
Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions
Annu. Rev. Biochem.
(2005)
Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene
Mol. Cell. Biol.
Maintenance and modulation of T cell polarity
Nat. Immunol.
Abnormal development of urogenital organs in Dlgh1-deficient mice
Development
Discs-large homolog 1 regulates smooth muscle orientation in the mouse ureter
Proc. Natl. Acad. Sci. USA
Differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus-associated lesions as a marker for progression to malignancy
Int. J. Cancer
Cited by (13)
Defining the in vivo mechanism of air pollutant toxicity using murine stress response biomarkers
2023, Science of the Total EnvironmentApplication of the in vivo oxidative stress reporter Hmox1 as mechanistic biomarker of arsenic toxicity
2021, Environmental PollutionCitation Excerpt :Supernatant was recovered and protein concentrations were measured by Bradford assay (Biorad). SDS-PAGE and immunoblotting was carried out as previously described (Inesta-Vaquera et al., 2010) with minor modifications. Antibodies used included hmox1 (Abcam, ab13243), β-gal (Promega, Z3781), NQO1 (Ab2346), GAPDH (Cell signaling, 2118).
Discovery of potent and selective covalent inhibitors of JNK
2012, Chemistry and BiologyCitation Excerpt :Despite this plethora of compounds, many exhibit poor kinase selectivity and/or do not inhibit the phosphorylation of well-characterized substrates of JNK in cells. For example, one of the earliest and still most widely used inhibitors is the anthrapyrazolone, SP-600125 (Bennett et al., 2001; Figure 1A), which exhibits exceptionally low specificity for JNK (Bain et al., 2007) and should only be used in combination with other tools to rule out a potential role for JNK in a particular process (Iñesta-Vaquera et al., 2010). Other reported JNK inhibitors such as AS601245 (Gaillard et al., 2005) only inhibit c-Jun phosphorylation at high concentrations, which is likely due to a combination of limited cell penetration, ATP concentration, and differences between biochemical and cellular sensitivities to JNK inhibitors.
CDK1, not ERK1/2 or ERK5, is required for mitotic phosphorylation of BIM <inf>EL</inf>
2012, Cellular SignallingCitation Excerpt :Thus, the ERK1/2 pathway seems not to be involved in mitotic phosphorylation of BIMEL. ERK5 is phosphorylated during mitosis and following treatment with nocodazole or paclitaxel [13,14,39,40] (Fig. 1A and B). It has been suggested that ERK5 directly phosphorylates BIMEL to promote survival during mitotic arrest [14].
Abnormal distribution of hDlg and PTEN in premalignant lesions and invasive cervical cancer
2011, Gynecologic OncologyCitation Excerpt :The HR-HPV E6 proteins bind to the PDZ-2 domain of the tumor suppressor protein hDlg (human homologue discs large) [8]. hDlg is a member of the membrane-associated guanylate kinase (MAGUK) protein family, which is involved in the inhibition of cell cycle progression [9,10]. The E6–hDlg interaction leads to the ubiquitination and degradation of hDlg by the proteosome pathway [11].
ERK5 signalling pathway is a novel target of sorafenib: Implication in EGF biology
2021, Journal of Cellular and Molecular Medicine