PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3

Abstract The phosphatases of regenerating liver (PRLs) are involved in tumorigenesis and metastatic cancer yet their cellular function remains unclear. Recent reports have shown PRL phosphatases bind tightly to the CNNM family of membrane proteins to regulate magnesium efflux. Here, we characterize...

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Autores principales:	Huizhi Zhang, Guennadi Kozlov, Xinlu Li, Howie Wu, Irina Gulerez, Kalle Gehring
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0fb93675d99f4329802dc664671cbeb7
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spelling	oai:doaj.org-article:0fb93675d99f4329802dc664671cbeb72021-12-02T16:06:09ZPRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM310.1038/s41598-017-00147-22045-2322https://doaj.org/article/0fb93675d99f4329802dc664671cbeb72017-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-00147-2https://doaj.org/toc/2045-2322Abstract The phosphatases of regenerating liver (PRLs) are involved in tumorigenesis and metastatic cancer yet their cellular function remains unclear. Recent reports have shown PRL phosphatases bind tightly to the CNNM family of membrane proteins to regulate magnesium efflux. Here, we characterize the interactions between the CBS-pair (Bateman) domain of CNNM3 and either PRL2 or PRL3 using X-ray crystallography, isothermal titration calorimetry, and activity assays. We report four new crystal structures of PRL proteins bound to the CNNM3 CBS-pair domain that reveal the effects of cysteine disulphide formation and nucleotide binding on complex formation. We use comprehensive mutagenesis of the PRL3 catalytic site to quantify the importance of different PRL amino acids, including cysteine 104, leucine 108, and arginine 110, for CNNM binding and phosphatase activity. We show the PRL3 R138E mutant is selectively deficient in CNNM3 binding with the potential to distinguish between the downstream effects of phosphatase and CNNM-binding activities in vivo. Through a novel activity assay, we show that PRL3 has magnesium-sensitive phosphatase activity with ATP and other nucleotides. Our results identify a strong correlation between phosphatase activity and CNNM binding and support the contention that PRL function as pseudophosphatases regulated by chemical modifications of their catalytic cysteine.Huizhi ZhangGuennadi KozlovXinlu LiHowie WuIrina GulerezKalle GehringNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Huizhi Zhang Guennadi Kozlov Xinlu Li Howie Wu Irina Gulerez Kalle Gehring PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3
description	Abstract The phosphatases of regenerating liver (PRLs) are involved in tumorigenesis and metastatic cancer yet their cellular function remains unclear. Recent reports have shown PRL phosphatases bind tightly to the CNNM family of membrane proteins to regulate magnesium efflux. Here, we characterize the interactions between the CBS-pair (Bateman) domain of CNNM3 and either PRL2 or PRL3 using X-ray crystallography, isothermal titration calorimetry, and activity assays. We report four new crystal structures of PRL proteins bound to the CNNM3 CBS-pair domain that reveal the effects of cysteine disulphide formation and nucleotide binding on complex formation. We use comprehensive mutagenesis of the PRL3 catalytic site to quantify the importance of different PRL amino acids, including cysteine 104, leucine 108, and arginine 110, for CNNM binding and phosphatase activity. We show the PRL3 R138E mutant is selectively deficient in CNNM3 binding with the potential to distinguish between the downstream effects of phosphatase and CNNM-binding activities in vivo. Through a novel activity assay, we show that PRL3 has magnesium-sensitive phosphatase activity with ATP and other nucleotides. Our results identify a strong correlation between phosphatase activity and CNNM binding and support the contention that PRL function as pseudophosphatases regulated by chemical modifications of their catalytic cysteine.
format	article
author	Huizhi Zhang Guennadi Kozlov Xinlu Li Howie Wu Irina Gulerez Kalle Gehring
author_facet	Huizhi Zhang Guennadi Kozlov Xinlu Li Howie Wu Irina Gulerez Kalle Gehring
author_sort	Huizhi Zhang
title	PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3
title_short	PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3
title_full	PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3
title_fullStr	PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3
title_full_unstemmed	PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3
title_sort	prl3 phosphatase active site is required for binding the putative magnesium transporter cnnm3
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/0fb93675d99f4329802dc664671cbeb7
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_version_	1718385069156990976

PRL3 phosphatase active site is required for binding the putative magnesium transporter CNNM3

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