Translational Control of C-terminal Src Kinase (Csk) Expression by PRL3 Phosphatase

Fubo Liang,Yong Luo,Yuanshu Dong,Chad D Walls,Jiao Liang,Hao-Yuan Jiang,Jeremy R Sanford,Ronald C Wek,Zhong-Yin Zhang

doi:10.1074/jbc.m708285200

Abstract

Phosphatase of regenerating liver 3 (PRL3) is up-regulated in cancer metastases. However, little is known of PRL3-mediated cellular signaling pathways. We previously reported that elevated PRL3 expression increases Src kinase activity, which likely contributes to the increased tumorigenesis and metastasis potential of PRL3. PRL3-induced Src activation is proposed to be indirect through down-regulation of Csk, a negative regulator of Src. Given the importance of PRL3 in tumor metastasis and the role of Csk in controlling Src activity, we addressed the mechanism by which PRL3 mediates Csk down-regulation. PRL3 is shown to exert a negative effect on Csk protein synthesis, rather than regulation of Csk mRNA levels or protein turnover. Interestingly, the preferential decrease in Csk protein synthesis is a consequence of increased eIF2 phosphorylation resulting from PRL3 expression. Reduced Csk synthesis also occurs in response to cellular stress that induces eIF2 phosphorylation, indicating that this regulatory mechanism may occur in response to a wider spectrum of cellular conditions known to direct translational control. Thus, we have uncovered a previously uncharacterized role for PRL3 in the gene-specific translational control of Csk expression.

Highlights

(7), breast [8], ovarian [9], and melanoma [7]
Our results suggest that elevated Phosphatase of regenerating liver 3 (PRL3) expression leads to a reduction in C-terminal Src kinase (Csk) protein synthesis, by a mechanism involving the eIF2 kinase pathway
We discovered that elevated PRL3 expression causes a reduction in Csk protein, leading to Src activation

Summary

EXPERIMENTAL PROCEDURES

Real-time Quantitative RT-PCR Analysis—The Csk mRNA level was determined by a two-step quantitative RT-PCR protocol using the fluorescent intercalating dye SYBR-Green RTPCR kit (Invitrogen) and an ABI Prism 7700 sequence detection system (Applied Biosystems). Cell lysates were cleared by centrifugation at 15,000 rpm for 15 min. 10 ␮g of antibody was added to 1 mg of cell lysate and incubated at 4 °C for 2 h. Pulse Chase Experiment—Cells were grown to 70% confluency, treated with methionine- and cysteine-free DMEM supplemented with dialyzed 10% fetal bovine serum for 30 min. Denatured Csk and ␤-actin probes were added to the membrane filters for 8 h at 60 °C with the presence of 100 ␮g/ml salmon sperm DNA in 1ϫ SSC buffer (15 mM NaCl, 15 mM sodium citrate, pH 7.0). Membrane was wrapped in cling-film, and radioactive signals were detected by exposing membrane to film for 24 h at Ϫ70 °C

RESULTS

To ensure that the observed

DISCUSSION