Abstract
Ran (Ras-related nuclear) protein, a member of the Ras superfamily of GTPases, is best known for its roles in nucleocytoplasmic transport, mitotic spindle fiber assembly, and nuclear envelope formation. Recently, we have shown that the overexpression of Ran in fibroblasts induces cellular transformation and tumor formation in mice (Ly, T. K., Wang, J., Pereira, R., Rojas, K. S., Peng, X., Feng, Q., Cerione, R. A., and Wilson, K. F. (2010) J. Biol. Chem. 285, 5815-5826). Here, we describe a novel activated Ran mutant, Ran(K152A), which is capable of an increased rate of GDP-GTP exchange and an accelerated GTP binding/GTP hydrolytic cycle compared with wild-type Ran. We show that its expression in NIH-3T3 fibroblasts induces anchorage-independent growth and stimulates cell invasion, as well as activates signaling pathways that lead to extracellular regulated kinase (ERK) activity. Furthermore, Ran(K152A) expression in the human mammary SKBR3 adenocarcinoma cell line gives rise to an enhanced transformed phenotype and causes a robust stimulation of both ERK and the N-terminal c-Jun kinase (JNK). Microarray analysis reveals that the expression of the gene encoding SMOC-2 (secreted modular calcium-binding protein-2), which has been shown to synergize with different growth factors, is increased by at least 50-fold in cells stably expressing Ran(K152A) compared with cells expressing control vector. Knocking down SMOC-2 expression greatly reduces the ability of Ran(K152A) to stimulate anchorage-independent growth in NIH-3T3 cells and in SKBR3 cells and also inhibits cell invasion in fibroblasts. Collectively, our findings highlight a novel connection between the hyper-activation of the small GTPase Ran and the matricellular protein SMOC-2 that has important consequences for oncogenic transformation.
Highlights
Given that the ability of Ran to cycle between its GDP- and GTP-bound states is linked to proper nucleocytoplasmic transport in cells, we wanted to examine mutants of Ran capable of accelerated GDPGTP exchange, within the context of cell growth control and cellular transformation
Given that we had previously shown that EGF message levels were increased in cells expressing the activated Ran(F35A) mutant and that EGFR signaling was a necessary component of cellular transformation by Ran(F35A) [13], we examined whether inhibiting EGFR tyrosine kinase activity with AG1478 blocked the up-regulation of SMOC-2 expression in Ran(K152A)
We reported that serum, as well as specific growth factors such as heregulin, which binds to ErbB3 and ErbB4 and activates ErbB2 tyrosine kinase activity, increase the pool of GTP-bound Ran in cells [13]
Summary
Results: Here, we describe a novel activating mutant of Ran and how it up-regulates the expression of the matricellular protein SMOC-2 and induces oncogenic transformation. We describe a novel activated Ran mutant, Ran(K152A), which is capable of an increased rate of GDP-GTP exchange and an accelerated GTP binding/GTP hydrolytic cycle compared with wild-type Ran. We show that its expression in NIH-3T3 fibroblasts induces anchorage-independent growth and stimulates cell invasion, as well as activates signaling pathways that lead to extracellular regulated kinase (ERK) activity. Knocking down SMOC-2 expression greatly reduces the ability of Ran(K152A) to stimulate anchorage-independent growth and cell invasion Overall, these findings highlight a previously unappreciated role for Ran in cellular growth regulation that is dependent on the matricellular protein SMOC-2
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