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

✍ Scribed by Sharon M. Barr; Edward M. Johnson

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 639 KB
• Volume: 81
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.1099

✦ Synopsis

Abstract

Levels of Purα, 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 Purα is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Purα 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 Purα can affect colony formation and anchorage‐independent growth in ras‐transformed NIH3T3 cells. Two to five‐fold elevated levels of Purα in stably‐transfected cell lines retard entry into and progression through S phase in both ras‐transformed and non‐transformed cells. Purα significantly inhibits colony formation by ras‐transformed cells but not by non‐transformed cells. In addition, cells transfected to express Purα 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 Purα. Biochemical analysis of nuclear and cytoplasmic Purα proteins and confocal microscopic analysis of Purα location indicate that access of Purα to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Purα domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215–231. In the nucleus Purα colocalizes with CDK2 and cyclin A. Purα and cyclin D1, however, do not colocalize in the nucleus. At mitosis Purα is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Purα 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. © 2001 Wiley‐Liss, Inc.