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J. Biol. Chem., Vol. 283, Issue 32, 22304-22315, August 8, 2008

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G2E3 Is a Dual Function Ubiquitin Ligase Required for Early Embryonic Development^*

William S. Brooks¹, E. Scott Helton, Sami Banerjee, Melanie Venable^¶, Larry Johnson^||, Trenton R. Schoeb^||, Robert A. Kesterson^||, and David F. Crawford²

From the Departments of Cell Biology, ^||Genetics, Pediatrics, and ^¶School of Medicine, University of Alabama, Birmingham, Alabama 35233

G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for mitotic regulatory proteins. It shuttles between the cytoplasm and nucleus, concentrating in nucleoli and relocalizing to the nucleoplasm in response to DNA damage. In this study, we demonstrate that G2E3 is an unusual ubiquitin ligase that is essential in early embryonic development to prevent apopotic death. This protein has a catalytically inactive HECT domain and two distinct RING-like ubiquitin ligase domains that catalyze lysine 48-linked polyubiquitination. To address in vivo function, we generated a knock-out mouse model of G2E3 deficiency that incorporates a β-galactosidase reporter gene under control of the endogenous promoter. Animals heterozygous for G2E3 inactivation are phenotypically normal with no overt change in development, growth, longevity, or fertility, whereas G2E3 null embryos die prior to implantation. Although normal numbers of G2E3^-/- blastocysts are present at embryonic day 3.5, these blastocysts involute in culture as a result of massive apoptosis. Using β-galactosidase staining as a marker for protein expression, we demonstrate that G2E3 is predominantly expressed within the central nervous system and the early stages of limb bud formation of the developing embryo. In adult animals, the most intense staining is found in Purkinje cell bodies and cells lining the ductus deferens. In summary, G2E3 is a dual function ubiquitin ligase essential for prevention of apoptosis in early embryogenesis.

Received for publication, April 28, 2008 , and in revised form, May 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants P30 CA13148 (to the University of Alabama Comprehensive Cancer Center Core), P30 AR48311 (to the Rheumatic Diseases Core Center) and K08CA86941. This work was also supported grants from the Hope Street Kids Foundation and The Research Institute at Children's Hospital (Children's Hospital of Alabama (DFC)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Biology, Freed-Hardeman University, Henderson, TN 38340.

² To whom correspondence should be addressed: 940 Stanton L. Young Blvd., BMSB 311, Oklahoma University Health Science Center, Oklahoma City, OK 73104. Tel.: 405-271-8001, ext. 52655; Fax: 405-271-3756; E-mail: david-crawford{at}ouhsc.edu.

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