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NIDA Proteomics Center
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Investigators
> Richard P. Lifton
Phosphoproteomic Studies of
Neuronal WNK Kinases
Richard P. Lifton, Department of Genetics,
Yale University
Intracellular chloride levels, [Cl-]i, define whether the
neurotransmitter GABA is excitatory or inhibitory. This fundamental property is
essential to the neural circuitry of the mammalian brain. We aim to reveal how
phosphorylation of the WNK family of signaling kinases directly controls [Cl-]i
in neurons. In addition, we aim to identify novel proteins (genes) that control
the physiological set point of [Cl-]i in neurons; an
essential component of brain function where major players (protein) have yet to
be defined. These studies aim to identify fundamental molecular mechanisms that
modulate the normal brain physiology that is ultimately altered by drugs of
addiction. WNK kinases co-expressed in mammalian cells with their known targets,
proteins that control [Cl-]i levels in neurons, will serve
as a model system to identify phosphoproteins that modulate neuronal [Cl-]i.
The complexity of this system requires novel applications of phosphoproteomic
technology. Preliminary studies with WNK kinases and K-Cl co-transporters have
identified 14 phosphorylation sites. Investigating dynamics in this complex
system will require SILAC approaches, coupled to targeted phosphoproteomics.
Targeting phosphorylation dynamics with SILAC coupled to titanium dioxide based
proteomic technologies will pinpoint phosphorylation dynamics that modulate [Cl-]i
in response to alterations via physiologic stimuli or site directed mutagenesis
of target proteins. The results of the study will define novel signaling
mechanisms in the mammalian brain and thus contribute to the over all goals of
the NIDA Center. These studies are expected to serve as a template for current
and future investigations by other members of the NIDA Center. Achieving these
goals will serve to fill a knowledge gap in understanding the molecular
mechanisms in the brain that establish the normal physiology that is altered by
drugs of addiction. |
