Abstract
Erythroid Krüppel-like factor (EKLF; KLF1) is an erythroid-specific transcription factor required for the transcription of genes that regulate erythropoiesis. In this paper, we describe the identification of a novel EKLF interactor, Ppm1b, a serine-threonine protein phosphatase that has been implicated in the attenuation of NFκB signaling and the regulation of Cdk9 phosphorylation status. We show that Ppm1b interacts with EKLF via its PEST1 sequence. However, its genetic regulatory role is complex. Using a promoter-reporter assay in an erythroid cell line, we show that Ppm1b superactivates EKLF at the β-globin and BKLF promoters, dependent on intact Ppm1b phosphatase activity. Conversely, depletion of Ppm1b in CD34(+) cells leads to a higher level of endogenous β-globin gene activation after differentiation. We also observe that Ppm1b likely has an indirect role in regulating EKLF turnover via its zinc finger domain. Together, these studies show that Ppm1b plays a multilayered role in regulating the availability and optimal activity of the EKLF protein in erythroid cells.
Highlights
Erythroid Kruppel-like factor (EKLF) is a transcription factor that is critical for erythroid gene expression
Using a promoter-reporter assay in an erythroid cell line, we show that Ppm1b superactivates EKLF at the -globin and BKLF promoters, dependent on intact Ppm1b phosphatase activity
We show that EKLF interacts with Ppm1b, a protein phosphatase that belongs to the PP2C family of protein phosphatases [23], and we describe its ability to modulate EKLF function by various means, including regulation of EKLF protein stability and transcriptional function
Summary
EKLF is a transcription factor that is critical for erythroid gene expression. Results: The EKLF in vivo interaction with Ppm1b phosphatase has been identified after co-immunoprecipitation. MAY 4, 2012 VOLUME 287 NUMBER 19 transcription regulation (4 –11) These studies were driven by knowledge of the erythroid-specific transcription factors or coactivator proteins that compose the essential transcriptional machinery and were designed to find out if EKLF interacted with these known molecules. Given the restricted tissue specificity and temporal regulation of EKLF expression in vivo, it is possible that post-translational regulation and/or protein interactions govern which of the opposing roles EKLF plays in the cell divisions that lead to terminal erythroid differentiation. Given the circumstantial evidence in the literature, most compellingly the cytoplasmic localization of EKLF, we hypothesized that EKLF might interact with, or be regulated by, proteins that are not typically part of the transcription machinery To address this question, we immunoprecipitated FLAGtagged EKLF that was expressed in MEL cells and identified associated proteins using mass spectrometry. We show that EKLF interacts with Ppm1b, a protein phosphatase that belongs to the PP2C family of protein phosphatases [23], and we describe its ability to modulate EKLF function by various means, including regulation of EKLF protein stability and transcriptional function
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