Spry2 does not directly modulate Raf-1 kinase activity in v-Ha-ras-transformed NIH 3T3 fibroblasts

Abstract

Sprouty (Spry) proteins have previously been suggested as negative regulators of the MAPK pathway through interaction with Raf-1. However, the molecular basis of this inhibition has not been elucidated. In this study, we used cells expressing FLAGtagged Raf-1 with point mutations at known phosphorylation sites to reveal that activation of Raf-1 mutants does not correlate with their degree of interaction with Spry2. The association of Raf-1 with Spry2 in intact cells was further corroborated by immunofluorescence colocalization. Additionally, there was no significant change observed in the strength of interaction between Raf-1 mutants and Spry2 after paclitaxel treatment despite differences in the activation levels of these mutants. Thus, our study provides the evidence that Spry2 does not directly regulate Raf-1 kinase activity, but instead acts as a scaffolding protein that assists interactions between Raf-1 kinase and its direct regulators. [BMB reports 2010; 43(3): 205-211]

Decreased Interaction of Raf-1 with Its Negative Regulator Spry2 as a Mechanism for Acquired Drug Resistance

“…Our recent data indicate that Spry2 may regulate Raf-1 kinase activity by acting as a scaffolding protein that brings Raf-1 kinase into close proximity with its direct regulators (Ahn et al, 2010). Here, we revealed that PDGF-induced Raf-1 activation was inversely correlated with the strength of interaction with Spry2 as monitored by co-immunoprecipitation experiment (Fig.…”

“…In contrast to the detailed data collected on the Raf-1 activation process, the mechanism responsible for Raf-1 inactivation after signaling events is much less understood. Possible candidate for the feedback negative regulation of the Raf-1/MAP kinase pathway is the Sprouty (Spry) protein (Yusoff et al, 2002;Sasaki et al, 2003;Ahn et al, 2010), which was found to be a general inhibitor of receptor tyrosine kinases (Hacohen et al, 1998). Our study revealed that the expression level of Spry2 in Ras-NIH 3T3/Mdr cells was much higher than in their parental cells.…”

“…For immunofl uorescence experiments, cells were grown on chamber slides (Nunc), and fi xed in 10% formalin solution for 10 min as described (Ahn et al, 2010). Samples were blocked with blocking solution (1% BSA, 0.6% Triton X-100 in PBS) for 1 h and incubated with primary antibodies diluted in blocking solution for 16 h at 4 o C. FITC-coupled anti-mouse IgG was used to detect Raf-1 proteins while Texas Red-coupled antirabbit IgG was used to visualize Spry2.…”

Autophagy-Dependent Survival of Mutant B-Raf Melanoma Cells Selected for Resistance to Apoptosis Induced by Inhibitors against Oncogenic B-Raf

“…For immunofluorescence experiments, cells were grown on chamber slides (Nunc), and fixed in 10% formalin solution for 10 min as described (Ahn et al ., 2010). Samples were blocked with blocking solution (1% BSA, 0.6% Triton X-100 in PBS) for 1 h and incubated with primary antibodies diluted in blocking solution for 16 h at 4℃.…”

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene

“…Anticancer activity is induced by promoting tubulin polymerization and inhibiting microtubules disassembly (2,3). Therefore, at subnanomolar concentrations, paclitaxel can block mitosis and inhibit cell proliferation (4). Since the Food and Drug Administration's approval of paclitaxel in 1982, it has also been approved to treat refractory ovarian cancer, metastatic breast cancer (5), AIDS-related Kaposi's sarcoma (6), and non-small cell lung cancer in many countries (7).…”