Transcriptional Modification by a CASK-Interacting Nucleosome Assembly Protein

CASK, a synaptic membrane associated guanylate kinase (MAGUK) protein, acts as a coactivator for transcription factor Tbr-1. Tbr-1 is an essential T-box transcription factor in cerebral cortex development. Presently, the molecular mechanism of the CASK co-activation effect is unclear. Here, we report that CASK interacts with another nuclear protein, CINAP (CASK-interacting nucleosome assembly protein), which binds histones and facilitates nucleosome assembly. CINAP, via its interaction with CASK, forms a complex with Tbr-1, regulating expression of the genes controlled by Tbr-1 and CASK, such as NR2b. NR2b is an important subunit of NMDA receptor, which plays critical roles in neuronal responses and diseases. Over-expression of Tbr-1 and CASK enhances NR2b expression. A knock down of endogenous CINAP in hippocampal neurons reduces the promoter activity of NR2b. The results support that Tbr-1, CASK, and CINAP are important for NR2b expression. Moreover, NMDA stimulation results in a reduction in the level of CINAP protein, via a proteasomal degradation pathway, correlating with a decrease in NR2b expression in neurons. This study suggests that reduction of the CINAP protein level by synaptic stimulation contributes to regulation of the transcriptional activity of the Tbr-1CASK-CINAP protein complex and thus modifies expression of the NR2b gene. This provides a negative feedback mechanism underlying the down-regulation of NR2b expression by synaptic stimulation, which may protect neurons from excitotoxicity. Via controlling NR2b expression, Tbr-1, CASK, and Figure 1. Interaction of CINAP, CASK, and Tbr-1. (A) Schematic structure of mouse CINAP. Proline (P)-, arginine-, and lysine (RK)-rich regions, the nucleosome assembly protein (NAP) domain, and two PEST motifs of CINAP are marked as black boxes. (B) Coimmunoprecipitation of CASK and CINAP from transfected COS cells. (C) Coimmunoprecipitation of CASK and CINAP from rat brain. Nonimmune mouse IgG was used as a negative control. (D) Nuclear colocalization of CINAP, Tbr-1, and CASK in P1 rat brain. The images represent the same field visualized for CINAP (cy3), CASK (FITC), and Tbr-1 (cy5), as indicated. Scale bar, 10 m.