Effects of prenyl pyrophosphates on the binding of S-Ras proteins with KSR.
BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang et al., 2001. J. Exp. Zool. 289:441-448). On a Western blot, the kinase suppressor of Ras (KSR) in the membrane fraction was expressed at slightly reduced level as compared to that of the untransformed cells. To understand this in more detail, the interaction of the bacterially expressed shrimp Ras (S-Ras) with KSR was investigated using KSR purified from mice brains. SDS-polyacrylamide gel electrophoresis and Western blot analysis revealed that the monomers of the purified KSR have a relative molecular mass of 60,000. Purified KSR was found to bind with digoxigenylated S-ras-encoding fusion protein (Dig-S-Ras) with high affinity in the absence of ATP, and the binding activity of KSR was sustained upon phosphorylation of Dig-S-Ras with mitogen-activated protein kinase (MAPK). The association of purified KSR with S-Ras was confirmed. Differences between the effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate on the binding of S-Ras with the purified KSR were assessed. Densitometer analysis revealed that at nanogram concentration, farnesyl pyrophosphate inhibited the binding of S-Ras with KSR competently, but geranylgeranyl pyrophosphate did not. The present study provides the evidence that decrease of the concentration of farnesyl pyrophosphate to sub-microgram levels lower the affinity of Ras proteins with KSR in the signaling pathway.
Chen
Chau-Huei
CH
Division of Biochemistry and Molecular Sciences, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529.
Fan
Junn-Huei
JH
Chuang
Nin-Nin
NN
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D033364:Decapoda; D011756:Diphosphates / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012717:Sesquiterpenes; D014162:Transfection
D011756:Diphosphates; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D011494:Protein Kinases; C097199:KSR-1 protein kinase; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras); C002963:geranylgeranyl pyrophosphate