ABSTRACT
Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem-like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial-mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome-wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem-like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells in vivo highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.
Subject(s)
Glioblastoma/genetics , Glioblastoma/pathology , Guanine Nucleotide Exchange Factors/metabolism , Lymphoid Enhancer-Binding Factor 1/genetics , Wnt Signaling Pathway/genetics , Zinc Finger E-box-Binding Homeobox 1/genetics , Cell Movement/genetics , DNA-Binding Proteins/genetics , Epithelial-Mesenchymal Transition/genetics , Glioblastoma/mortality , Guanine Nucleotide Exchange Factors/genetics , Humans , Neoplasm Invasiveness/genetics , Transcription, Genetic/genetics , Transcriptional Activation/genetics , Zinc Finger E-box-Binding Homeobox 1/metabolismABSTRACT
Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.
Subject(s)
Carrier Proteins/metabolism , Cyclin-Dependent Kinase 5/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Hippocampus/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Amino Acid Substitution , Animals , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/metabolism , Carrier Proteins/genetics , Cells, Cultured , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 5/genetics , Guanine Nucleotide Exchange Factors/genetics , Hippocampus/cytology , Humans , Membrane Proteins/genetics , Mutation, Missense , Nerve Tissue Proteins/genetics , Neurons/cytology , Phosphorylation/physiology , Rats , Rho Guanine Nucleotide Exchange Factors , Synaptic Membranes/genetics , Synaptic Membranes/metabolismABSTRACT
Collybistin (Cb) is a brain specific guanine nucleotide exchange factor that interacts with the inhibitory postsynaptic scaffold protein gephyrin. Cb is essential for the postsynaptic clustering of gephyrin and major GABA(A) receptor subtypes during the formation and maintenance of GABAergic synapses in the hippocampus and other areas of the forebrain. In the rat, four distinct splice variants (Cb1, Cb2(SH3-), Cb2(SH3+) and Cb3), have been described, which differ in their C-termini (Cb1-3) and in respect of the SH3-domain that is absent in Cb2(SH3-). In the human brain, only a single isoform (hPEM2) corresponding to Cb3, was found to be expressed. This has been implicated in neurological defects such as hyperekplexia, epilepsy, anxiety, aggression and mental retardation. In this study, we address the functional significance of the differentially spliced Cb isoforms by generating a shRNA-mediated knock-down of endogenous Cb in hippocampal cultured neurons that is subsequently rescued by the expression of distinct Cb isoforms. We found that the Cb knock-down induced impairment in GABAergic neurotransmission could be rescued by the expression of any of the Cb isoforms, independent of their C-termini or the presence of the SH3-domain in the N-terminal region. Thus, the different Cb isoforms all confer basic functionality.
