The Ca2+ Channel β4c Subunit Interacts with Heterochromatin Protein 1 via a PXVXL Binding Motif

Xingfu Xu,Yoon J Lee,Johanna B Holm,Mark D Terry,Robert E Oswald,William A Horne

doi:10.1074/jbc.m110.187864

Xingfu Xu, Yoon J Lee + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.187864

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Abstract

The β subunits of voltage-gated Ca(2+) channels are best known for their roles in regulating surface expression and gating of voltage-gated Ca(2+) channel α(1) subunits. Recent evidence, however, indicates that these proteins have a variety of Ca(2+) channel-independent functions. For example, on the molecular level, they regulate gene expression, and on the whole animal level, they regulate early cell movements in zebrafish development. In the present study, an alternatively spliced, truncated β4 subunit (β4c) is identified in the human brain and shown to be highly expressed in nuclei of vestibular neurons. Pull-down assays, nuclear magnetic resonance, and isothermal titration calorimetry demonstrate that the protein interacts with the chromo shadow domain (CSD) of heterochromatin protein 1γ. Site-directed mutagenesis reveals that the primary CSD interaction occurs through a β4c C-terminal PXVXL consensus motif, adding the β4c subunit to a growing PXVXL protein family with epigenetic responsibilities. These proteins have multiple nuclear functions, including transcription regulation (TIF1α) and nucleosome assembly (CAF1). An NMR-based two-site docking model of β4c in complex with dimerized CSD is presented. Possible roles for the interaction are discussed.

Highlights

Be related to the high levels of ␤4 expression in yolk syncytial nuclei [4]
Sequence comparisons led to the initial discovery that Ca2ϩ channel ␤ subunits are membraneassociated guanylate kinase proteins consisting of core Src homology 3 and guanylate kinase (GK)2 domains connected by a large variable loop (HOOK) [6]
Using multiple in vitro biochemical and biophysical techniques, we show that ␤4c interacts with the chromo shadow domains (CSDs) of heterochromatin protein 1 (HP1) dimers and that the interaction occurs via two ␤4c sites, the primary of which is a C-terminal PXVXL consensus sequence

Summary

Introduction

Be related to the high levels of ␤4 expression in yolk syncytial nuclei [4]. Other studies have shown that ␤ subunit functions in the nucleus may be cell type- and splice variant-specific [5]. For the first set of titration experiments, NMR protein samples, 15N-labeled ␤4c⌬199 or ␤4c⌬184 and unlabeled CSD, were dissolved in buffer containing 50 mM HEPES, 100 mM NaCl, 50 mM Glu/Arg, 10% D2O, pH 7.0.

Results

Conclusion