Received for publication July 15, 2005.
Revised September 22, 2005.
Accepted for publication October 5, 2005.

REGULATION OF CXCR4-MEDIATED NUCLEAR TRANSLOCATION OF ERK1/2

Abstract

CXCR4 activation has been associated with cell migration and proliferation in many cell types, but the intracellular signaling cascades are incompletely defined. Here we show that CXCR4-dependent ERK1/2 phosphorylation was mediated through the Ras/Raf pathway as demonstrated with a dominant negative Ras mutant and pharmacological inhibitors. The Src inhibitor, PP1, and the Rho-kinase (ROCK) inhibitor, Y27632, also attenuated SDF-1 induced ERK1/2 activation. Involvement of Src could furthermore be demonstrated by Src phosphorylation and by the shortened ERK1/2 phosphorylation in SYF cells, which are Src/Yes/Fyn deficient compared to Src reconstituted Src⁺⁺ cells. Membrane translocation of RhoA could similarly be detected. A large portion of the SDF-1-mediated ERK phosphorylation was detected in the nucleus, as shown by Western blotting and confocal microscopy, and resulted in the phosphorylation of the transcription factor Elk. Interestingly, the nuclear accumulation of ERK1/2 and Elk phosphorylation was completely blocked by DN-Rho, Y27632, PP1 and latrunculin B, indicating that the Rho/Rock pathway, Src kinase and the actin cytoskeleton were required in this process. In accordance, neither nuclear ERK activation nor Elk phosphorylation were observed in SYF cells stimulated with SDF-1, but were reconstituted in Src⁺⁺ cells. In summary, these results demonstrate that Src, Rho/ROCK and an intact cytoskeleton contribute to overall ERK1/2 activation in SDF-1 stimulated cells, and are indispensable for nuclear translocation of ERK1/2 and activation of transcription factors.

Key words: Chemotactic peptides, Src and other nonreceptor tyrosine kinases, Cdc42, rho, rac, other small G proteins, MAP Kinase

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