.IEEE INDIA ANNUAL CONFERENCE 2004. LNDlCON 2004
401,
Raf Kinase Inhibitor Protein: A Putative Molecular
Target in Prostate Cancer
Shailza Singh,B.K.Malik, D.K:Sharma
Abstract: Each year in the United States, prostate cancer lrills
over 40,000 men, and over 200,000 new case? are dhgnosed,
making it the second most common cancer and the second most
common cause of cancer deaths among males. Wnfortunatefy,
the best available diagnostic tests are substantially less than 100
percent sensitive and specific, and many men have incurable
prostate cancer at the time of diagnosis which created the need
for the development of new drugs to reduce prostate cancer risk.
Raf kinase inhibitor protein (RKIP), a member of the
phosphatidyl ethanolamine binding protein (PEBP) family, is a
small cytosolic protein associated with signal hausdudon
pathways. RKIP is a suppressor of metastases, whlcb paved the
way for structural and functional efforts towards drug
development; here we describe the molecular modeling of RKlP
that should provide a structural framework on which the design
of speciflc inhibitors may be based.
Keywords: RaCkinase;Bioinfonnatifs;Structure;Drug design
Cancer can be considered a disease of communication at the
molecular leve1. Prostate cancer is the single most common
form of solid tumor in humans and is among the most
common malignancies. Current estimates indicate that 1 in 6
men in the United States will be diagnosed with prostate
cancer in his lifetime, in 2002, an estimated 189,000 prostate
cancer diagnoses were made in the United States,
accompanied by estimated 30.200 prostate cancer deaths.
(Reiter RE,deKemion JB ZOOZ), Despite the high prevalence
and clinical importance of prostate cancer, the primary cause
or causes and the mo1ecula.r mechanisms underlying the
development and progression of this disease is poorly
understood. The lack of knowledge about these mechanisms
is among the most important reasons why there are no
effective prevention strategies or treatment modalities to cure
advanced prostate cancer.
Biochemistry has played an ancillary role in elaborating the
mechanistic aspects of the interactions. However, an
understanding of their biochemistry becomes paramount
when we are challenged to define potential targets for drugs
and to devise suitable screening systems for drug discovery.
Bioinfor~naticsis a powerful tool and is a new paradigm in
the detection and diagnosis of prostate cancer.
Shailza Singh
.
Center for Energy Studies, Indian Institute of Technology,
H a w Khas. New Delhi-1 100L6.Phone No.01 1-26591256
Fax: +91-11-26582077,
e-rrail:shailza_iitd@yahoo~com ,
esr01021 @ccsun50.iitd.emet.in
BXMalik and DX. S h a m , Institute of Genomics and Integrative
Biology,CSIR, Mall Road.Delhi-1loo07
.
1
The underlying cause of death lies in the progression and
metastasis of the primary cancer, Defining the mechanisms
that confer metastatic ability on cancer cells is an important
goal towards prevention of metastasis. (Rinker-Schaeffer
CW, Welch DRYSokoloff M..2000). Analysis of metastatic
prostate cancers has identified the RKIP as a suppressor of
metastasis. A gene array screen between a non-metastatic
prostate cancer cell and its metastatic derivative line revealed
decreased expression of Raf kinase inhibitor protein (RKIP)
in the metastatic cell line. (Fu,Z.et al.2003). This finding is
consistent with the possibility that loss of RKIP is associated
with metas'tasis. RKIP is expressed in many tissues including
brain, lung, and liver. RKIP blocks Raf-induced
phosphorylation of MEK. (Yeung K, Janosch P, McFerran B,
Rose DW, Mischak H, Sedivy JM, Kolch W., 2000). In
addition to its modulation of Raf signaling, RKIP modulates
both G-protein signaling and NF-kappa activity. (Yeung
KC, Rose DW, Dhillon AS, Yaros D, Gustafsson M,
Chatterjee D, McFerran B, Wyche J, Kolch W, Sedivy
JM.,2001)XRKIppromotes apoptosis of cancer cells, which
suggests that loss of RKIP in cancer will protect cancer cells
against cell death. Additionally, restoration of RKIP
expression in metastatic prostate cancer cell line does not
affect primary tumor growth, but it does inhibit prostate
cancer metastasis. These parameters identify RKIP as a
metastasis suppressor gene, which suggest that it or proteins
it interacts with are putative molecular targets to control
metastasis. These findings are supported by the observation
that RKIP expression is decreased in metastases of prostate
cancer patients, compared to normal prostate or the primary
prostate tumor, Previous studies demonstrated that RKIP
binds to Raf-1 and prevents the activation of the extracellular
regulated kinase (ERK) cascade. ( Yeung K, Seitz T,Li S,
Janosch P, McFerran B, Kaiser C, Fee F, Katsanakis KD,
Rose DW, Mischak H, Sedivy JM, Kolch W.1999). New
work shows that phosphorylation of RKIP by protein kinase
C disassociates RKIP from Raf-1 and stimulates its binding
to, and inhibition of G-protein-coupied receptor kinase 2
(GRK2). (Krasel, C. et d.2001). This switching enhances
signaling by activation of the ERK pathway and by decreased
receptor desensitization. ((3x1R,Baldassare JJ, 2004). Using
hydrophobic
cluster
analysis
and
molecular
modeling,Schoentgtn et al. showed that the bovine RIUP
(Robinson L.C et aI, 1991)may possess a potential nucleotide
binding site and suggested that it may belong to the kinase
family and promote the transfer of hydrophobic ligands to the
plasma membrane.(Schoentgen F et a1.1992).
The present paper describes the molecular model of human
Raf kinase inhibitor protein (RKIP)..Thehomology modeling
0-7803-8909-3/04/$20.00020M IEEE.
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407
INDIAN 1NSTlTUTE OF TECHNOLOGY, KHARAGPUR 721302, DECEMBER 20-22,2004
was performed using crystallographic structure of Raf lcinase
inhibitor protein from Bos tau-, solved to resolution better
than 2.6A' as template. The results presented here provide a
three-dimensional model of Euup to aid the design of
specific inhibitors.
Methods
Secondary structure prediction. The secondary structure was
predicted by
using
PSIPRED
and
PHD
(Profile
Network from
PHD results (brief)
SYNUPSIS of prediction
PHDsec summary overall prc-:in c n be la sified
as :
mixed given the following classes:
o - 'all-alpha': %H ;45% AND %E < 5%
o 'all-beta': %H < 5% AND %E > 45%
o 'alpha-beta': %H > 30% AND %E > 20%
o 'mixed': all others
Hiedelberg).
Molecular Modeling. For modeling of the RKIP we used
restrained-based modeling implemented h the program
MODELLER.(A. Sali & T.L.Blundell., 1993). This program
is an automated approach to comparative modeling by
satisfaction of spatial restraints. The modeling procedure
begins with an alignment of the sequence to be modeled
(target) with related known three-dimensional structures
(templates). This alignment is usually the input to the
program. The output is a three-dimensional model for the
target sequence containing all min-chainand side-chain nonhydrogen atoms.
The degree of primary sequence identity between RKIP and
Bos laurus RKIP (btRKIP)indlcates hat the crystallographic
structure ofbtRKIP (PDB access code: la44) is a good model
to be used as template for FWIP.
Predicted secondary structure composition for
RKIP protein:
Predicted solvent accessibiIity composition
(cordsurface ratio) for RKIP protein:
Classes used:
o e: residues exposed with more than 16% of
their surface
o b: all other residues.
A~ialysisofthe model. The overall stereochemical quality of
the final model for RKIP was assessed by the program
PROCHECK.(Laskowski R A, MacArthur M W, Moss D S
& Thornton J M.1993). .
,,
Active site prediction. The active site was predicted by using
WHATIF SERVER (Vriend.,'G. 1990 ) with FlexX.where Raf
Kinase Inhibitor
Protein (RKIP) PDB file was uploaded and the resulting
pockets were obtained.
44.62
GLOBE prediction of globularity says that protein appears
to be compact and has a globular domain.
Rcsults and Discussion
Secmdmy structure prediction. The secondary structure
'
55.38
was
predicted by using PSIPRED.(McGuffm U, Bryson K, Jones
DT, 2000) PSIPIED incorporates two feed-forward n e m l
networks, which perform an analysis on output obtained from
PSI-BLAST (Position Specific Iterated BLAST)(Altschul et
al., 1997). The average prediction accuracy for PSIPRED in
three structural states is 76.5%, which is higher than any of
the other secondary structure prediction methods. Neural
network method (PHD)(B Rost and J Li~,2003) correctly
predicts 82.2% of residues for 74% of co-predicted amino
acids.
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1EEE INDIA ANNUAL CONFERENCE 2004,I?IDiCON 2004
408
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Primary Sequence comparison
The sequence alignment of btRKIP (template) from and
RKZP (target) is shown in Fig.1. The sequence RKlp shows
94.1% of identity with the sequence of btRKIP.
Fig.2. Molecular Modef of K I P from MODELLER
Qualip of the model
Figs3A and B show the Ramachandran diagram a-$plots for
the human RKIP structure and for crystallographic btRKIP
stnrcture solved to resolution better than 2.6A'. The
Ramachandran plot for the btRKIP was generated to compare
the overall stereochemical quality of human RKIP model
against RKIP structures solved by biocrystallography.
Analysis of the Ramachandran. Plot of the human RKlP
model shows that 94.8% of the residues lie in the most
favored regions and the remaining 5.2% in the addtional
allowed regions. The same analysis for the crystaliographic
btRKIP structure present 91.4% of residues in the most
favorable and 8.6% in the additional allowed regions. The
overall rating for the human RKIP model is good.
mi b l = m i
Fig.3B Ramachandran Plot of btRKIP [pdb la44)
Active Site
The active site of an enzyme is the region that binds the
substrate and contributes the amino acid residues that directly
participate in the making and breaking of chemical bonds.
Active site is a 3-dimensional entity, not a point or a plane
usually an intricate pocket or cleft structurally designed to
accept the structure of the substrate in 3-D terms. Protein
active sites (Fig.4A) usually contain water molecules that,
upon ligand binding, are either displaced into bulk solvent or
retained to mediate protein-ligand interactions.. The
contribution of water molecules to ligand binding must be
accounted for to compute accurate values of binding
affinities. This requires estimation of the extent of hydration
of the binding site. However, it is often difficult to identify
the water molecules involved in the binding process when
ligands bind on the surface of a protein.Binding pockets of
raf kinase inhibitor protein is shown in Fig.4B.
Pocket
-l
Fig.4A Pockets of Raf Kinase Inhibitor protein
Fig.4B Binding Pockets'of ATP-binding site Raf Kinase
Inhibitor protein
Fig.3A Ramachandran Hot of K I P
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409
INDIAN INSTITUTE OF TECHNOLOGY, KHARAGPUR 721302, DECEMBER 20-22,2004
ConcIusions
C.Hotredame, D.Higgins, J. Heringa(2000) "T-Coffee: A novel methcd for
multiple sequence alignments." Journal ojMolecularBiulogy. 302,205-217.
Fu, 2. et al. (2003)Effects of Raf kinase inhibitor protein expression on
suppression of prostate cancer metastasis. J. Nd.cancer fmt.95,878-889.
Goel R, Baldassare JJ. (2004)Killing two birds with one RKIP. Trends
molecular, cellular, physiological, and pathological events
EndocrinoI Metab.15(3):91-92.
that lead to uncontrolled growth and metastasis. In this work
Kfllcr ET,.Fu 2,Yeung K, Brennan M.(2004)Raf kinase inhibitor protein: a
prostate cancer metastasis suppressor gene. :Cancer Lett. 207(2):1 3 1 137.
we described the homology modeling on RKIP:
Krasel, C. et al. (2001) Phosphorylation of GRK.2 by protein kinase C
supplementary studies will allow us to shed new light on the
abolishes its inhibition by calmodulin.J.Biol.CIrem.276,l911-1915.
binding features of this protein, which cannot be crystallized , Laskowski R A , MacArthur M W.Moss D S & Thornton J M (1993).
easily. There are still many challenging problems in prostate , PROCHECK a program lo check the stereochemical quality of protein
cancer and the related research is very active, This ongoing
Structures. J. Appl. CYS~.,
26,283-291.
McGuffin U. Bryson K, Jones DT. (2000) The PSIPRED protein structure
research effort has provided a wealth of new scientific
prediction server. Bioinformo!icr. 16,404405.
opportunities that if pursued may further advance our
Reiter RE,d e h i o n JB (2002). Epidemiology, etiology, and prevention of
understanding of prostate cancer. We believe, with the
prostate cancer. In PC Walsh et al., eds., Campbett's Urology, 8th ed., vol. 4,
pp. 3003-3024.Philadelphia: W.B. Saunders.
technical improvement in modeling methods and so many
Rinker-Schaeffer CW, Welch DR, SokoloffM.(2000)Defining the biologic
genes (protein sequences) discovered, protein-modeling tools
role
of genes that regulate prostate cancer melastasis. Curr Opin Urof.fO(5):
will play an even more important role in the post-genome era
397401.
and this is an opportune time to reassess and plan a research
Robinson LC,Tatchell K.(1991)TFSI: a suppressor ofcdc25 mutations in
agenda for prostate cancer that will guide the field into the
Saccharomyces cerevisiue,Mol.Gen.Gencl:(230):24 I -250.
Schoentgen F,Seddiqi N,Bucquoy SJolfes P,lemesleVarloot L,provost
next decade, With the aid of molecular modeling, 12-0KMomon J.P.(1992) Main structural and functional features of the basic
tetradecanoylphorbol-13-acetate (TPA)(a pyrrolidone
cytosolic bovine 2 I kDa protein delineated through hydrophobic cluster
analogue) could serve as a modulator to develop novel drugs
analysis and molecular modeling.Protein Engng(5) 295-303.
to treat prostate cancer. Further in silico work has to be
Vknd.,G. WHATIF: A molecular modeling and drug design program.
J. Mol. Gruph. 1990;8,52-56.
performed in order to confirm this prediction.
Ycung K,Janosch P, McFwran E,Rose DW, Mischak H,Sedivy JM,Kolch
W.(2000) Mechanism of suppression of the RaWEWextracellular signalReferences:
regulated kinase pathway by the mf kinase inhibitor protein. :Mol Cell B i d .
Altschul SFMadden TLSchaffer A4,Zhmg J,ZhanB &Milla W,Lipmon
20(9): 3079-3085.
DJgappcd BLAST and PSI-BUSTh ncw generation of protein database
Yeung K, Seitz T,Li S,Janosch P, McFerran B,Kaiser C, Fee F, Katsanakis
search program. Nucleic Acids Res. 1497;25:3389-3402.
KD, Rose DW, Mischak H, Sedivy JM,Kolch W. (1999)Suppression of
A. Sali & T.L. Btundell. (1993)Comparative protein mudelling by
Raf-1 kinase activity and MAP kinase signalling by W:Nature
satisfaction of spatial restraints. J. Mol. B i d . 234,'779-815.
173-177.
B Rost and J Liu (2003) Predicthtein: ?%he PredicPmieinSaver. Nucleic 401(6749):
Yeung KC. Rose DW, Dhillon AS,Yams D. Gustafsson M,Chattejee D,
Acidr Research 3/(13): 330G3304.
McFerran B, Wyche J, Kolch W, Sedivy JM. (2001)Raf Kinase lnhibitor
C. Geoujon & G. Deleage (1994) A brief description is given in: "SOPM :a
Protein Interacts with NPxB-Inducing Kinase and TAKl and Inhibits NFself optimised prediction mcthcd for protein secondary structure prediction."
KBActivation :Mol Cell B i d (21): 7207Protein &ngineering. 7, 157-164.
In summary, to iinprove diagnosis, prevention, and treatment
of prostate cancer, it is crucial to focus future research on the
-
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