Biochemical and Biophysical Research Communications
FLJ23654 encodes a heart protein phosphatase 1-binding protein (Hepp1)
Introduction
Protein phosphatase 1 (PP1), a major serine/threonine protein phosphatase, is associated in vivo with a family of regulatory subunits that target it to different subcellular locations and modulate its activity toward specific substrates [1], [2], [3], [4] Most of the PP1 regulatory proteins contain a consensus PP1-binding motif, (R/K)(V/I)X(F/W), which binds to the surface of the enzyme distant from the active site [5], [6], [7], [8]. More than 50 PP1 regulatory proteins have been identified [9], but several lines of evidence indicate that a number of novel PP1 regulatory proteins remain to be identified [10], [11], [12]. In a previous study, we employed the yeast two-hybrid method to identify several clones that encoded novel PP1 regulatory proteins [13]. One of them was phostensin, encoded by KIAA1949, which targets PP1 to the F-actin cytoskeleton. In the current study, we report that FLJ23654 encodes a novel PP1-binding protein, which we have termed Hepp1 for heart protein phosphatase 1-binding protein. Hepp1 mRNA was exclusively expressed in heart and testis, and enhanced the activity of PP1, suggesting it may play an important role in cardiac function.
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Materials and methods
Reagents. Tris, LB broth, DTT, ampicillin, phenylmethylsulfonyl fluoride, benzamidine and imidazole were obtained from Sigma. Thrombin, GSH-Sepharose, blue-Sepharose and metal chelating-Sepharose were obtained from Amersham Biosciences. PP1 and PP2A were prepared as described [14]. Phospho-inhibitor-1 and inhibitor-2 were prepared as described [15]. Proteins extracted from human heart tissues were obtained from Clontech.
5′-rapid amplification of cDNA ends (5′-RACE). 5′-RACE was performed using
Identification of PP1 regulatory proteins
We conducted a search for proteins capable of interacting with PP1 in human heart using a yeast two-hybrid screen. Eight distinct classes of cDNAs were obtained from 39 positive clones. Twenty-two positive clones were from one unique class, suggesting that the heart contains an abundance of this class of cDNAs. DNA sequencing analysis indicated that these 22 clones were the product of the gene FLJ23654. We then used 5′-RACE analysis to determine the 5′ sequence of the FLJ23654 transcript
Acknowledgments
This work was supported by grants from the National Science Council, ROC (NSC 94-2320-B-194-005 and NSC 95-2320-B-194-005) and the Taipei Veterans General Hospital, Taiwan, ROC (VGH 92-375, VGH 93-359 and VGH 94-370).
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Regulating the regulator: Insights into the cardiac protein phosphatase 1 interactome
2016, Journal of Molecular and Cellular CardiologyCitation Excerpt :The second interactor was named Hepp1 for heart PP1-binding protein and its mRNA was found exclusively in heart and testis by Northern blot [46]. When the catalytic activity of PP1α was probed in the presence of I-1 and I-2, Hepp1 antagonized the inhibition of both phospho-I-1 and I-2 and non-significantly increased PP1α's activity on its own [46]. Using PP1 co-immunoprecipitation (IP) followed by MS, we also found an interaction between PP1 and phostensin in both mouse ventricles and human atria but did not detect Hepp1 [9].
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These authors contributed equally to this work.