Phospholipid Scramblase 1 Binds to the Promoter Region of the Inositol 1,4,5-Triphosphate Receptor Type 1 Gene to Enhance Its Expression

Quansheng Zhou,Iris Ben-Efraim,Jo-Lawrence Bigcas,Daniela Junqueira,Therese Wiedmer,Peter J Sims

doi:10.1074/jbc.m504821200

Quansheng Zhou, Iris Ben-Efraim + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m504821200

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Abstract

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, endofacial membrane protein originally identified based on its capacity to promote accelerated transbilayer phospholipid movement in response to Ca(2+). Recent evidence suggests that this protein also participates in cell response to various growth factors and cytokines, influencing myeloid differentiation, tumor growth, and the antiviral activity of interferon. Whereas plasma membrane PLSCR1 was shown to be required for normal recruitment and activation of Src kinase by stimulated cell surface growth factor receptors, PLSCR1 was also found to traffic into the nucleus and to tightly bind to genomic DNA, suggesting a possible additional nuclear function. We now report evidence that PLSCR1 directly binds to the 5'-promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene (IP3R1) to enhance expression of the receptor. Probing a CpG island genomic library with PLSCR1 as bait identified four clones with avidity for PLSCR1, including a 191-bp fragment of the IP3R1 promoter. Using electrophoretic mobility shift and transcription reporter assays, the PLSCR1-binding site in IP3R1 was mapped to residues (-101)GTAACCATGTGGA(-89), and the segment spanning Met(86)-Glu(118) in PLSCR1 was identified to mediate its transcriptional activity. The significance of this interaction between PLSCR1 and IP3R1 in situ was confirmed by comparing levels of IP3R1 mRNA and protein in matched cells that either expressed or were deficient in PLSCR1. These data suggest that in addition to its role at the plasma membrane, effects of PLSCR1 on cell proliferative and maturational responses may also relate to alterations in expression of cellular IP3 receptors.

Highlights

Multiply palmitoylated, Ca2ϩ-binding, Pro- and Cys-rich, endofacial plasma membrane protein that was shown to distribute into lipid raft domains and to be a substrate of the Abl and Src tyrosine kinases (4 – 6)
The exact biologic function of this protein remains controversial; Phospholipid scramblase 1 (PLSCR1) mediates Ca2ϩ-dependent transbilayer movement of PL in proteoliposomes [2, 3] and was reported to increase cell surface expression of phosphatidylserine through remodeling of plasma membrane PL in mammalian cells exposed to Ca2ϩ ionophore and other inducers of injury or apoptosis [7,8,9], it has been observed that induced elevation of PLSCR1 expression can occur without a detectable increase in PL movement between membrane leaflets, and gene deletion of PLSCR1 did not impair cell capacity to undergo this remodeling of cell surface PL (10 –12)
Whereas the PLSCR(1&3)Ϫ/Ϫ kidney epithelial cells (KEC) used in these experiments were matched in both GFP fluorescence and in expressed mRNA, the level of expression of the PLSCR1-C3 A&K3 A double mutant was always distinctly less than that of the WT or PLSCR1-C3 A constructs (Fig. 7C), potentially reflecting accelerated degradation of the double mutant construct in the cytosol. These experiments identify a gene that is directly transcriptionally regulated by PLSCR1, a multiply palmitoylated plasma membrane protein that was recently discovered to traffic into the nucleus as cargo of the importin nucleopore chaperones and to bind directly to DNA

Summary

Introduction

Multiply palmitoylated, Ca2ϩ-binding, Pro- and Cys-rich, endofacial plasma membrane protein that was shown to distribute into lipid raft domains and to be a substrate of the Abl and Src tyrosine kinases (4 – 6). Our data suggest that PLSCR1 binds to a segment of the 5Ј-promoter of IP3R1, enhances transcription of this gene, and is required for normal expression of cell IP3R1 protein.

Results

Conclusion