ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

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Abstract

Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

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).

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