Dataset Information

The Ca2+ channel beta4c subunit interacts with heterochromatin protein 1 via a PXVXL binding motif.

ABSTRACT: The ? 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 ?(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 ?4 subunit (?4c) 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 1?. Site-directed mutagenesis reveals that the primary CSD interaction occurs through a ?4c C-terminal PXVXL consensus motif, adding the ?4c subunit to a growing PXVXL protein family with epigenetic responsibilities. These proteins have multiple nuclear functions, including transcription regulation (TIF1?) and nucleosome assembly (CAF1). An NMR-based two-site docking model of ?4c in complex with dimerized CSD is presented. Possible roles for the interaction are discussed.

SUBMITTER: Xu X

PROVIDER: S-EPMC3058963 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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The Ca2+ channel beta4c subunit interacts with heterochromatin protein 1 via a PXVXL binding motif.

Xu Xingfu X Lee Yoon J YJ Holm Johanna B JB Terry Mark D MD Oswald Robert E RE Horne William A WA

The Journal of biological chemistry 20110110 11

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 ...[more]

PMID: 21220418

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Project description:L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of μ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-μ1A and the GST-μ1A C-terminal domain, but not the GST-μ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357, 359KK360, and 362KK363) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (369DD370) in the membrane-distal end of the L-selectin tail involved in μ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-μ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of μ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo Molecular docking of the L-selectin tail to μ1A was used to identify the μ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates μ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin.

Dataset Information

The Ca2+ channel beta4c subunit interacts with heterochromatin protein 1 via a PXVXL binding motif.

Publications

The Ca2+ channel beta4c subunit interacts with heterochromatin protein 1 via a PXVXL binding motif.

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