Abstract
c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family. It has become clear that JNK does not only have a role in induction of stress responses but also in processes such as cell movement. In this report we demonstrate that JNK activity is necessary for platelet-derived growth factor (PDGF)-BB-induced chemotaxis of primary foreskin fibroblasts and in other cell types. PDGF-BB stimulation was found to lead to activation of JNK with a maximum after 30 min. Inhibition of JNK reduced Ser178 phosphorylation of the focal adhesion component paxillin. Paxillin phosphorylation at this site has been shown to be involved in the dynamics of focal adhesions and consequently cell migration. Moreover, we observed localization of JNK to the actin-dense membrane ruffles induced by PDGF-BB stimulation both using immunofluorescence staining and green fluorescent protein-tagged JNK. This suggests a role for JNK at the leading edge of the cell compatible with a function in cell migration. Furthermore, we show that phosphatidylinositol 3-kinase (PI 3-kinase), which has an established role in PDGF-stimulated cell migration, is necessary for PDGF-induced activation of JNK. In conclusion, JNK is a critical component downstream of PI 3-kinase that may be involved in PDGF-stimulated chemotaxis presumably by modulating the integrity of focal adhesions by phosphorylating its components.
Highlights
Jun N-terminal kinase (JNK) has frequently been suggested to have essential roles in inflammation, differentiation, and apoptosis (7, 8)
We have demonstrated that PDGF-BB stimulation results in Ser178 phosphorylation of paxillin in a JNKdependent manner
We propose that JNK may be a down-stream effector of PI 3-kinase important for PDGF-induced cell migration, possibly through paxillin Ser178 phosphorylation
Summary
Reagents—Recombinant human PDGF-BB was generously provided by Amgen (Thousand Oaks, CA) and STI571 by Novartis Pharma AG (Basel, Switzerland). Cells were washed and incubated in Ham’s F-12 medium containing 0.1% fetal bovine serum. Immunoprecipitations and Western Blotting—Subconfluent AG01523 cells were starved as described above and incubated for 1 h with inhibitors at the indicated concentrations and thereafter stimulated with 20 ng/ml PDGF-BB for the indicated periods of time. For Western blotting, samples were electro-transferred to polyvinylidene difluoride membranes (Immobilon P), which were blocked in 5% milk powder (anti-phospho-JNK and anti-phospho-c-Jun) or 1% bovine serum albumin (anti-phospho-paxillin) in phosphate-buffered saline solution containing 0.1% Tween 20. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (both from Amersham Biosciences), and proteins were visualized using ECL Western blotting detec-. Cells were blocked by incubation for 1 h in 10 mM glycine, pH 7.4, at room temperature, and incubated with 5% fetal bovine serum and JNK antibody overnight at 4 °C. Coverslips were mounted on glass slides using Fluoromount-G (Immunkemi, Sweden), and to visualize the fluorescence, a Zeiss microscope was used
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