Open AccessSpry2 does not directly modulate Raf-1 kinase activity in v-Ha-ras-transformed NIH 3T3 fibroblasts
Author(s) -
Jun Ho Ahn,
Ki Hwan Eum,
Michael Lee
Publication year - 2010
Publication title -
bmb reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.511
H-Index - 77
eISSN - 1976-670X
pISSN - 1976-6696
DOI - 10.5483/bmbrep.2010.43.3.205
Subject(s) - mapk/erk pathway , colocalization , mutant , microbiology and biotechnology , protein kinase a , immunoprecipitation , chemistry , kinase , phosphorylation , scaffold protein , signal transduction , biology , biochemistry , gene
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.