SCIENTIFIC REPORT 2004 - Sylvester Comprehensive Cancer Center

T U M O R C E L L B I O L O G Y P R O G R A M
the membrane-bound second messenger leads to
the catalytic activation of their respective kinase
domains.
A combination of high-resolution heteronuclear
multidimensional nuclear magnetic resonance
(NMR) methods, nuclear Overhauser
effects, and nuclear relaxation rates will be used
to determine the effects that Ins(1,3,4,5)P 4
binding
has on the solution structures and dynamics
of the bacterially expressed recombinant 15 N- and
13
C-isotopically labeled PH domain constructs of
both human PDK1 and PKB/Akt. In addition,
the recombinant 15 N-isotopically labeled PH domain
constructs of both PDK1 and PKB/Akt will
be spliced with their corresponding bacterially
expressed recombinant unlabeled kinase domains
to determine the effects that Ins(1,3,4,5)P 4
binding
to the PH domain has on the conformations,
dynamics, and position of the PH domain with
respect to the corresponding kinase domain.
Finally, the modes of activation of PDK1
and PKB/Akt will be elucidated by measuring the
effects of Ins(1,3,4,5)P 4
binding to the PH domains
on the equilibrium and activation free energies
associated with binding of nucleotide,
metal, or protein substrates, conformational
changes, and covalent catalysis. Such structural
and mechanistic understanding will be useful in
the rational design of potent and selective inhibitors
by “linking” the free energies of binding of
substrate analogs with analogs of the inositol polar
head group of the phospholipid second messenger.
SELECTED PUBLICATIONS
2002
Harris, TK and Turner, GJ. Structural basis of
perturbed pKa values of catalytic groups in enzyme
active sites. IUBMB Life 53:85-98, 2002.
2003
Harris, TK . PDK1 and PKB/Akt: ideal targets
for development of new strategies to structurebased
drug design. IUBMB Life 55:117-26,
2003.
Gao, X, Yo, P, Keith, A, Ragan, TJ, and Harris,
TK. Thermodynamically balanced inside-out
(TBIO) PCR-based gene synthesis: a novel
method of primer design for high-fidelity assembly
of longer gene sequences. Nucleic Acids Research
31:e143, 2003.
HIGHLIGHTS/DISCOVERIES
• Designed and synthesized codon-optimized
genes and gene constructs for PDK1 and PKB/
Akt in order to optimize production of 15 N-
and 13 C-isotopically labeled human PDK1 and
PKB/Akt necessary for NMR structural and
dynamical studies, which facilitate high-level
protein production in bacteria. Researchers developed
a novel thermodynamically balanced
inside-out (TBIO) method of polymerase chain
reaction (PCR)-based synthesis to generate the
codon-optimized human kinase genes.
• Filed a U.S. Patent and Trademark Office provisional
patent application for the TBIO method
on August 28, 2003.
MARY LOU KING, PH.D.
Professor of Cell Biology and Anatomy
DESCRIPTION OF RESEARCH
Dr. King is trying to understand how spatial
patterning and cell fate is determined in the
early Xenopus embryo. Researchers in her laboratory
and others in the field have shown that the
first step in patterning the embryo appears to be
the localization of specific mRNAs to the vegetal
cortex during oogenesis. These maternal mRNAs
are subsequently inherited by a subset of cells in
the embryo. Evidence indicates that the proteins
encoded by localized mRNAs influence gene expression
in a region-specific manner, leading to
cellular diversification. They are actively pursuing
the mechanism through which the spatial distribution
of mRNAs is established and maintained.
Dr. King’s laboratory has isolated seven localized
mRNAs from Xenopus oocytes. Remarkably,
three RNAs, Xcat-2 (related to nanos), Xdazl (in
UM/Sylvester Comprehensive Cancer Center Scientific Report 2004 77