Effects of prenyl pyrophosphates on the binding og S-ras proteins
with KSR
Introduction: Ras proteins are membrane-associated small guanine
nucleotides binding proteins.
In mam-mals, four isoforms of Ras exist:H-Ras,N-Ras,KA -Ras,
and KB -Ras.
These are products of three genes.KA -Ras and KB -Ras are splice
variants of the same gene. Among them,the protein sequences are 80%
identical with major differences residing in their carboxyl
termini,including the CAAX (C,cysteine; A,an aliphatic amino
acid)motifs of prenylation. Ras is modified by prenylation to increase its
intrinsic affinity for the plasma membranes at which they participate
in signal transduction.The addition of isoprenoid groups,such as
geranylger-anyl (20-carbon)and farnesyl groups (15-carbon),
is determined by the X residue of the carboxyl terminal CAAX of
proteins.
If X is leucine or phenylalanine,the protein is geranylgeranylated, if X is
methione, serine, alanine, or glutamine, the protein is farnesylated .All
mamma-lian Ras proteins are prenylated by protein farnesyltransferase.
However,KB -Ras is the only exception.KB –Ras can be acylated by
protein geranylgeranyltrans-ferase I (PGGTase I). The reason for the
existence of two types of prenylation in cells is not clear.
The kinase suppressor of Ras (KSR)has been genetically identified as an
important mediator of Ras-dependent signals either upstream of or
parallel to Raf in Drosophila melanoga-ster and Caenorhabditis
elegans.Expression of small amounts of KSR cooperates with Ras for
Xenopus oocyte maturation,whereas a high level of expression results in
Grant sponsors.
Mammalian forms of KSR-1 have been identified on the basis of
sequence homology.A catalytic role for KSR has been suggested by a
report that Thr 269 is the major c-Raf-1 site activated and phosphorylated
by KSR. On the other hand,a growing body of evidence indicates a
scaffolding role for KSR in the mitogen-activated protein kinase
(MAPK)pathway.KSR has been shown to interact with Raf-1,MEK,and
MAPK in co-immunopreci-pitation experiments and yeast two-hybrid
assays.
Problem: What are the effects of geranylgeranyl pyrophosphate compare
to farnesyl pyr-ophosphate on the binding of Ras with KSR.
Solution: the reserchers used a recombi-nant ras-encoded p25 fusion
protein of shrimp Penaeus japonicus (S-Ras)which shares 85%
homology with mammalian KB -Ras protein. S-Ras is prenylated
exclusively at CAAX by PGGTase I of shrimp but acylated by PFTase of
mammals as well.
cells were transformed by transfection with DNA encoding the mutated
ras(Q61 K)from shrimp Penaeus japonicus.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 SRas 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.
Summery:
The mutant S-Ras(Q61 K)protein has been shown to transform
mammalian BALB/3T3 cells.the results showed that farnesyl
pyrophosphate at sub-micro-gram levels inhibited the recruitment of SRas with KSR,but geranylgeranyl pyrophosphate did not.These findings
indicate that the prenylation with farnesyl pyrophosphate at the C-termini
of shrimp Ras provides a mechanism to exhaust the available amounts of
farnesyl pyrophosphate,and Ras is routed away from KSR and is diverted
to aberrant growth,such as transformation.