Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Purα in NIH3T3 cells

被引:48
作者
Barr, SM
Johnson, EM
机构
[1] Mt Sinai Sch Med, Dept Pathol, New York, NY 10029 USA
[2] Mt Sinai Sch Med, Dept Biochem & Mol Biol, New York, NY 10029 USA
关键词
PURA; ras; colony formation; focus formation; CDK; cyclin; PEST sequence;
D O I
10.1002/jcb.1099
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Levels of Pur alpha, a conserved, sequence-specific single-stranded DNA and RNA binding protein, fluctuate during the cell cycle, declining at the onset of S-phase and peaking at mitosis. In early G1 Pur alpha is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Pur alpha into NIH3T3 cells arrests the cell cycle at either G1/S or G2/M checkpoints with distinct morphological consequences. Here we ask whether expression of Pur alpha can affect colony formation and anchorage-independent growth in ras-transformed NIH3T3 cells. Two to five-fold elevated levels of Pur alpha in stably-transfected cell lines retard entry into and progression through S phase in both ras-transformed and non-transformed cells. Pur alpha significantly inhibits colony formation by ras-transformed cells but not by non-transformed cells. In addition, cells transfected to express Pur alpha formed only about 1/5 the number of large colonies in soft agar as control-transfected cells, demonstrating a marked inhibition of anchorage-independent growth by Pur alpha. Biochemical analysis of nuclear and cytoplasmic Pur alpha proteins and confocal microscopic analysis of Pur alpha location indicate that access of Pur alpha to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Pur alpha domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215-231. In the nucleus Pur alpha colocalizes with CDK2 and cyclin A. Pur alpha and cyclin D1, however, do not colocalize in the nucleus. At mitosis Pur alpha is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Pur alpha to interact with proteins regulating cell proliferation and transformation is controlled by signals that govern its intracellular localization. J. Cell. Biochem. 81:621-638, 2001. (C) 2001 Wiley-Liss. Inc.
引用
收藏
页码:621 / 638
页数:18
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