Identification of Three Major Sentrinization Sites in PML

doi:10.1074/jbc.273.41.26675

Identification of Three Major Sentrinization Sites in PML*

From the Division of Molecular Medicine, Department of Internal Medicine, Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas-Houston Health Science Center, Houston, Texas 77030

Abstract

Acute promyelocytic leukemia arises following a reciprocal chromosome translocation t(15;17), which generates PML-retinoic acid receptor α fusion proteins (PML-RARα). We have shown previously that wild type PML, but not PML-RARα, is covalently modified by the sentrin family of ubiquitin-like proteins (Kamitani, T., Nguyen, H. P., Kito, K., Fukuda-Kamitani, T., and Yeh, E. T. H. (1998) J. Biol. Chem. 273, 3117–3120). To understand the mechanisms underlying the differential sentrinization of PML versus PML-RARα, extensive mutational analysis was carried out to determine which Lys residues are sentrinized. We show that Lys⁶⁵ in the RING finger domain, Lys¹⁶⁰ in the B1 Box, and Lys⁴⁹⁰ in the nuclear localization signal contributes three major sentrinization sites. The PML mutant with Lys to Arg substitutions in all three sites is expressed normally, but cannot be sentrinized. Furthermore, the triple substitution mutant is localized predominantly to the nucleoplasm, in contrast to wild type PML, which is localized to the nuclear bodies. Thus, sentrinization of PML, in the context of the RING finger and the B1 box, regulates nuclear body formation. Furthermore, we showed that sentrinization of PML-RARα could be restored by overexpression of sentrin, but not by retinoic acid treatment. These studies provide novel insight into the pathobiochemistry of acute promyelocytic leukemia and the sentrinization pathway.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant HL-45851 (to E. T. H. Y.), the DREAM project (to E. T. H. Y.), an American Heart Association Established Investigator Award (to E. T. H. Y.), and an Arthritis Foundation Irene Dugan Arthritis Investigator Award (to T. K.). This is publication number 132-IMM from the Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas-Houston Health Science Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Dr. Hans Müller-Eberhard.
↵‡ To whom correspondence should be addressed: Division of Molecular Medicine, Dept. of Internal Medicine, The University of Texas-Houston Health Science Center, 6431 Fannin, Suite 4.200, Houston, TX 77030. Tel.: 713-500-6660; Fax: 713-500-6647; E-mail:eyeh{at}heart.med.uth.tmc.edu.
↵2 L. Gong and E. T. H. Yeh, manuscript in preparation.
Abbreviations:

APL

acute promyelocytic leukemia

RAR

retinoic acid receptor

RA

retinoic acid

HA

hemagglutinin

PBS

phosphate-buffered saline

HRP

horseradish peroxidase

nls

nuclear localization signal.
- Received February 25, 1998.
- Revision received May 29, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.