Spry2 does not directly modulate Raf-1 kinase activity in v-Ha-ras-transformed NIH 3T3 fibroblasts.
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.
Ahn
Jun Ho
JH
Department of Biology, College of Natural Sciences, University of Incheon, Incheon 406-840, Korea.
Eum
Ki Hwan
KH
Lee
Michael
M
D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; C118389:Spry2 protein, mouse; D018631:ras Proteins