phosphothreonine and Glioblastoma

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α (also known as EPAS1), are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin 2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma, ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates Thr27 of ID2, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumour growth, and a more favourable outcome for patients with glioblastoma.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cell Line, Tumor; Cullin Proteins; Dyrk Kinases; Glioblastoma; Humans; Hypoxia-Inducible Factor-Proline Dioxygenases; Inhibitor of Differentiation Protein 2; Male; Mice; Neoplastic Stem Cells; Oxygen; Phosphorylation; Phosphothreonine; Protein Binding; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Ubiquitination; Von Hippel-Lindau Tumor Suppressor Protein; Xenograft Model Antitumor Assays

Although PTEN (phosphatase and tensin homologue deleted on chromosome 10) is one of the most commonly mutated tumour suppressors in human cancers, loss of PTEN expression in the absence of mutation appears to occur in an even greater number of tumours. PTEN is phosphorylated in vitro on Thr366 and Ser370 by GSK3 (glycogen synthase kinase 3) and CK2 (casein kinase 2) respectively, and specific inhibitors of these kinases block these phosphorylation events in cultured cells. Although mutation of these phosphorylation sites did not alter the phosphatase activity of PTEN in vitro or in cells, blocking phosphorylation of Thr366 by either mutation or GSK3 inhibition in glioblastoma cell lines led to a stabilization of the PTEN protein. Our data support a model in which the phosphorylation of Thr366 plays a role in destabilizing the PTEN protein.

Topics: Animals; Casein Kinase II; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycogen Synthase Kinase 3; Humans; Mice; Mutation; NIH 3T3 Cells; Phosphorylation; Phosphothreonine; PTEN Phosphohydrolase; Serine

Protein phosphorylation and dephosphorylation is one of the main mechanisms of cell cycle regulation. This study examines the modulation of epidermal growth factor receptor phosphorylation as cells emerge from quiescence and enter the S phase of the cell cycle. The epidermal growth factor receptor is phosphorylated primarily on serine and threonine, but not on tyrosine residues, in an S phase-dependent fashion, as determined by phosphoamino acid analysis and anti-phosphotyrosine immunoblotting. These phosphorylations occur both in vitro and in vivo and are ligand independent. Some of the sites that are phosphorylated in vitro also appear to be phosphorylated in vivo, as determined by two-dimensional tryptic phosphopeptide analysis. At least one of the in vivo phosphorylation sites is phosphorylated by mitogen-activated protein kinase. Although the mechanism for this ligand-independent phosphorylation is not known, its correlation with emergence from quiescence and entry into the cell cycle suggests that the phosphorylation of epidermal growth factor receptor on serine and threonine residues may have heretofore unknown role(s) in cell cycle entry and progression.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Culture Media, Serum-Free; Electrophoresis, Gel, Two-Dimensional; ErbB Receptors; Flow Cytometry; Glioblastoma; Humans; Interphase; Phosphopeptides; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; S Phase; Tyrosine