The β Subunit of Voltage-gated Ca2+ Channels Interacts with and Regulates the Activity of a Novel Isoform of Pax6

Yun Zhang,Yoichi Yamada,Mingming Fan,Saroja D Bangaru,Bochao Lin,Jian Yang

doi:10.1074/jbc.m109.022236

Abstract

Ca(2+) channel beta subunits (Ca(v)betas) are essential for regulating the surface expression and gating of high voltage-activated Ca(2+) channels through their interaction with Ca(2+) channel alpha(1) subunits. In efforts to uncover new interacting partners and new functions for Ca(v)beta, we identified a new splicing isoform of Pax6, a transcription factor crucial for the development of the eye, nose, brain, and pancreas. Pax6 contains two DNA binding domains (paired domain and homeodomain), a glycine-rich linker connecting these two domains and a C-terminal proline-, serine-, and threonine-rich transactivation domain. The protein sequence and function of Pax6 are highly conserved from invertebrate to human. The newly isolated isoform, named Pax6(S), retains the paired domain, linker, and homeodomain of Pax6, but its C terminus is composed of a truncated classic proline, serine, and threonine domain and a unique S tail. Pax6(S) shows a similar level of transcriptional activity in vitro as does Pax6, but only in primates is the protein sequence highly conserved. Its spatial-temporal expression profiles are also different from those of Pax6. These divergences suggest a noncanonical role of Pax6(S) during development. The interaction between Pax6(S) and Ca(v)beta is mainly endowed by the S tail. Co-expression of Pax6(S) with a Ca(2+) channel complex containing the beta(3) subunit in Xenopus oocytes does not affect channel properties. Conversely, however, beta(3) is able to suppress the transcriptional activity of Pax6(S). Furthermore, in the presence of Pax6(S), beta(3) is translocated from the cytoplasm to the nucleus. These results suggest that full-length Ca(v)beta may act directly as a transcription regulator independent of its role in regulating Ca(2+) channel activity.

Highlights

Pax6 is a transcription factor that belongs to the paired box (Pax) family (16 –25)
Fusion of the Pax6 PST with the transcription factor GAL4 increases the activity of GAL4 [37, 39, 40], suggesting that the transactivity of the PST domain can be independent of the paired domain (PD) and HD
The protein encoded by No 8 contains the last 12 amino acids of the PD, the linker region, the HD, and the N-terminal half of the PST domain (PSTN) present in the canonical Pax6 plus a novel C-terminal tail encoded by exon 11␣ (Fig. 1B)

Summary

Introduction

Pax6 is a transcription factor that belongs to the paired box (Pax) family (16 –25). It is widely expressed in the eye, nose, pancreas, and the central nervous system in both embryonic and adult mammals, and it plays important roles in regulating the development of these tissues and organs (18, 20, 26 –31). The C-terminal PST domain plays a key role in regulating Pax6 transcriptional activity but does not bind DNA directly. The Pax6 consensus DNA binding sequence, CD19-2, was cloned into the promoter region of a modified pGL3-OFLuc vector (Promega) to produce the reporter construct.

Results

Conclusion