Glycogen-Synthase-Kinase-3 been linked to multiple human chronic diseases including;

Glycogen-Synthase-Kinase-3
(GSK3) is a ubiquitously expressed, protein kinase found in all eukaryotes and
exists in two isoforms; GSK3? and GSK3?. Evidence suggests that GSK3
phosphorylates over 100 substrates(1,2,3), with the potential for
many more still to be discovered. It acts as a downstream regulatory switch for
numerous signalling pathways, some of which include; cellular responses to WNT,
insulin, G Protein Coupled Receptors (GPCR) and receptor tyrosine kinases
(RTK). Consequently, GSK3 is involved in a huge variety of signal transduction
cascades and so, influences many cellular functions such as cell proliferation
and apoptosis, glucose regulation and cell cycle progression, just to name a
few. Due to its involvement in these signalling pathways GSK3 has been linked
to multiple human chronic diseases including; Alzheimer’s Disease, Cancer and
Diabetes. (4)

                GSK3? has been linked to Alzheimer’s
Disease pathobiology through its major role in the phosphorylation of tau
protein. Tau proteins act to stabilise microtubules however, when they become
hyperphosphorylated they consequentially fail to stabilise these microtubules,
and this leads to the development of nervous system diseases such as Alzheimer’s
Disease. In two studies by Forlenza, it was noted that patients with
Alzheimer’s Disease had increased GSK3 activity(5) and that Lithium
Chloride may be used as a preventive treatment due to the inhibition of GSK3?.(6)
The role of GSK3 in the Wnt signalling pathway has also been associated with
cancer progression. However, GSK3 functions differently dependent on the tumour
tissue type. In some tissue types GSK3 activity is diminished due to
phosphorylation by growth factors and this has been linked to cancer
progression. Alternatively GSK3 can, as previously discussed, regulate the cell
cycle and this is done via the Wnt/?-catenin signalling pathway.
If this pathway were to become de-regulated, this has been linked to the development
of breast cancer.(7) In addition GSK3 has been known to negatively
regulate proto-oncogenes and therefore, can be said to have tumour suppressant
functions.(8) In the mid-1990s GSK3 inhibition by insulin was found
to be crucial for insulin to enhance glycogen synthesis (glucose storage).
Insulin signalling, as well as growth factors, inhibit GSK3 activity by
phosphorylating Ser9 of GSK3 resulting in the blocked interaction between GSK3
and pre-phosphorylated (primed) substrates. Therefore, it could be considered
as a therapeutic target for diabetes. For example, if GSK3 is overexpressed or hyper
activated this would result in insulin resistance within type 2 diabetes. (4)
Diseases such as these, which are all linked to GSK3, occur at a high frequency
worldwide and effect patients of all ages. There is a global need for new and more
effective treatments to be discovered in order to combat these terrible,
life-threatening and altering diseases.

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                For these reasons GSK3 has been
proposed as a drug target, however, it is now known that GSK3 plays an
important role in fundamental cell physiology, hence the therapeutic window for
inhibition of this kinase is very narrow. Ideally, accurate biomarkers of GSK3
activity are required in order to establish and quantify the relationship
between disease and GSK3 activity and subsequently, to titrate GSK3 inhibitors
safely and effectively in vivo.

The
5 cell lines that were investigated include; rat hepatoma H4, human fibroblast
HEK293, mouse 3t3-L1, human neuroblastoma SHSY5Y and human lung cancer line
A549. The cell lines were treated with two GSK3 inhibitors; CT99021 (aka
CHIR99021) and Lithium Chloride (LiCl). CT99021 is a cell permeable compound
acting as an ATP competitive and highly specific inhibitor of GSK3 activity.
(9) Within animal models of disease, CT99021 has been found to increase
the power of insulin activation of glucose transport and utilisation, reducing
insulin resistance within muscle.(10) Lithium Chloride is a
selective inhibitor of GSK3 classically used as a mood stabiliser in the
ongoing treatment for Bipolar Disorder. In 1996 Klein and Melton (11)
discovered that Lithium Chloride directly inhibited GSK3 activity as well as
increased the inhibitory-phosphorylation of GSK3 resulting in an amplification
of the direct inhibitory action.(12) Within muscle and adipocyte
cell lines Lithium Chloride has a stimulatory effect on glycogen synthesis and
glucose transport. (10)

                In
order to measure GSK3 activity in cells or in vivo one can directly
immunoprecipitate GSK3 and assess the enzymatic activity towards a peptide
substrate per unit of cellular protein. This is a useful measure to have but is
technically challenging and time consuming. In addition, it only provides a
snapshot of the specific activity and represents the activity state of the
isolated enzyme towards a peptide substrate. This may not be representative of
the true enzyme activity present in multi-protein complexes, or of its activity
towards different substrates. Therefore an additional approach is to monitor
the phosphorylation of known GSK3 targets inside the cells or tissue of
interest. This provides a readout of GSK3 function, and includes the influence
of cellular location of GSK3 and phosphatases which would oppose any GSK3
action. The main issue with the approach is the availability of sensitive and
specific antibodies to validated phosphorylation sites on GSK3 targets, along
with the semi-quantitative nature of western blotting (which primarily allows
comparative studies rather than fully quantitative). To improve the approach,
it would be beneficial to have a panel of validated antibodies to specific GSK3
targets, a biomarker panel for GSK3 activity. My project aimed to investigate
the sensitivity and tissue selectivity of several antibodies to the proposed
GSK3 targets detailed below.

 

                The ?-catenin/Wnt
pathway regulates cell fate decisions and stem cell pluripotency during
development Wnt signalling also acts as a negative regulator of GSK3. When the
Wnt-signal is absent, ?-catenin is targeted for sequential GSK3 phosphorylation
by CK1 and the APC/Axin/GSK3?-complex. Within this complex, Axin acts as a
scaffolding protein binding GSK3 and ?-catenin in close proximity, allowing
for the phosphorylation of ?-catenin. This leads to the
ubiquitination and proteasomal degradation of ?-catenin. However, when Wnt is
present it binds to Frizzled (Fz) receptors resulting in the activation of
Dishevelled (Dsh). This processes leads to GSK3? being displaced from the
APC/Axin complex, thereby stabilising ?-catenin allowing transport to the
nucleus and the regulation of target gene transcription. ?-catenin is
associated with various cancers due to its role as a proto-oncogene. (13)
By this process we would expect to see an increase in ?-Catenin due to a
decrease in phosphorylation when GSK3 inhibitors are used. This has been shown
in previous studies through the use of CT99021 (10) and Lithium
Chloride, which recovered Wnt signalling via the inhibition of GSK3?, resulting
in ?-catenin stabilisation.(14) Therefore, changes in GSK3 activity
should associate with reciprocal changes in the total amount of ?-catenin in
the cell, hence total ?-catenin protein is a potential biomarker for GSK3
activity.

                Collapsin
response mediator protein 2 (CRMP2) is responsible for binding to microtubules
and regulating neuronal axon outgrowth. This is a process regulated via
sequential phosphorylation by GSK3 and cyclin-dependent kinase 5 (Cdk5), and
occurs specifically at sites that are hyperphosphorylated in Alzheimer Disease.
It is possible that either GSK3 or Cdk5 overexpression, or a decrease in CRMP2
phosphatase activity, would worsen the progression of Alzheimer’s disease, as
this would enhance CRMP2 and tau phosphorylation. In previous studies GSK3
inhibitors, such as CT99021, caused rapid dephosphorylation of CRMP2 at Thr-509(15),
and so this residue is a potential biomarker for GSK3 activity.

            The enzyme Glycogen Synthase is a central regulator of
the conversion of glucose into glycogen, contributing to the control of blood
glucose, by the dephosphorylation and activation of glycogen synthase. Glycogen
synthase was once considered the best known substrate of GSK3 as it was one of
the first to be discovered.(16) However, many more substrates have
been discovered since its first report in scientific literature in 1982. Casein
kinase-2 (CK2) phosphorylates glycogen synthase as a requirement to form the
recognition site for GSK3 phosphorylation, resulting in the reduction of
glycogen synthase activity. Insulin inhibits GSK3 thereby relieving the
inhibitory phosphorylation, promoting glycogen production and reducing blood
glucose. (9) This was the first GSK3 substrate reported and
represents a validated substrate (at least in muscle). Therefore the
phosphorylated glycogen synthase is a potential biomarker for GSK3 activity.

                Myc, a transcription factor, is
involved in the stimulation of protein translation via the enhancement of
expression of a variety of genes involved in the synthesis of nucleotides and
ribosomes.(13) When GSK3 is overexpressed this results in enhanced
phosphorylation, which facilitates the rapid proteolysis of C-Myc by the
ubiquitin pathway. In addition, GSK3 inhibition has been found to increase
C-Myc protein expression. (9) Therefore, total C-Myc protein is a
potential biomarker for GSK3 activity.

 

                Finally,
in addition to measuring these GSK3 targets my project aimed to assess total
GSK3 expression in each cell model (both GSK3 isoforms), along with assessing
the inhibitory phosphorylation of each GSK3 isoform (at Ser21 and Ser9 of GSK3?
and GSK3?
respectively). In this way it would be possible to assess whether there were any
differences in GSK3 isoform expression or regulation between the cell models.
In addition I would assess whether any action of the GSK3 inhibitors on the
biomarkers included alterations in the amount, or regulation, of either GSK3
isoform.