CRITICAL ROLE OF THE RING FINGER DOMAIN OF NP95, A NUCLEAR RING FINGER PROTEIN, IN TUMOR CELL GROWTH

Yonchu Jenkins*, Vadim Markovtsov, Betty Huang, Wayne Lang,
Michel Janicot, Jorge Vialard, Janine Arts, Thierry Grand-Perret, Donald Payan, and Yasumichi Hitoshi
Rigel, Inc., 1180 Veterans Blvd., South San Francisco, CA 94080,
USA and JJPRD, Beerse, Belgium.

INTRODUCTION.  Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or modulation of growth signaling pathways.  Identification of novel genes involved in cellular proliferation may lead to new therapeutic classes in treatment of cancer.  By screening a doxocycline-inducible GFP-fused cDNA library in A549 cells for genes that interfere with cell cycle progression, we have identified a fragment of Np95, a nuclear ring finger protein, that serves as a dominant negative effector of cell growth.  We present results describing the role of Np95 and its ring finger domain in human tumor cell proliferation.  

RESULTS.  One hit from our proliferation-based functional screen encoded a peptide corresponding to part of the G9a domain (amino acids 506 to 624) of NP95.  Overexpression of this fragment clearly exerted strong antiproliferative effects in several tumor lines.  To confirm that NP95 was required for tumor cell proliferation, we utilized siRNA-mediated NP95 message reduction.  Abrogation of NP95 expression using transient transfection of NP95-specific siRNAs in H1299 cells is antiproliferative and results in pronounced increases in the G1 and G2/M populations.  Sustained delivery of an NP95-specific siRNA using a retroviral vector leads to markedly enhanced growth defects in A549, HeLa, and H1299 cells.  Np95 has previously been shown to be transcriptionally downregulated upon G1 phase induction by serum starvation or cellular differentiation.^1,2  We find that treatment of A549 cells using drugs that activate different cell cycle checkpoints also results in differential downregulation of Np95 message levels, with a corresponding decrease in Np95 protein.  We have

performed dominant negative studies to analyze the role of the NP95 ring finger in cellular proliferation.  The primary sequence of NP95 contains a PHD and a          

ring finger domain, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that Np95 displays E3 ligase activity.  Mutation of the Np95 ring finger, a domain which is essential

for substrate ubiquitination in many ring finger type ligases, abrogates Np95 autoubiquitination.  We have analyzed the effects of overexpressing wild type Np95 and Np95 ring finger mutants on tumor cell proliferation.  As with the endogenous protein, GFP-fused Np95 wild type and ring finger mutants localize to the nucleus.  We found no obvious growth defects upon overexpression of GFP-fused NP95 ring finger mutants alone in A549 cells.  However, these cells display markedly enhanced sensitivity to a wide variety of chemotherapeutics, revealing a role for the ring finger domain in cellular proliferation. 

CONCLUSIONS.   Np95 was first identified as a novel murine nuclear protein whose expression was associated with proliferating cells³  although deletion of the NP95 gene in murine embryonic stem cells did not result in growth defects.⁴  Our results clearly indicate that NP95 plays an important role in human cell proliferation.  A fragment of NP95 was identified as a hit from a proliferation-based functional screen.  Reduction of NP95 message levels using NP95-specific siRNAs led to decreased growth rates in several tumor cell lines.  In addition, activation of a number of different cell cycle checkpoints resulted in downregulation of NP95.  We also show that NP95 has ubiquitin E3 ligase activity which is dependent upon the ring finger.  Overexpression of a GFP-fused Np95 ring finger mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics, implicating the ring finger domain of Np95 as a functional determinant of growth regulation.  Taken together, our results suggest a general requirement for NP95 in tumor cell proliferation. 

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