Protocols and Applications Guide (US Letter Size) - Promega

||||||| 7Cell Signaling
phosphorylation, including Bad, caspase-9 and GSK-3
(Cooray, 2004). Akt also regulates transcription of many
genes including forkhead transcription factors and NF-κB
(Cooray, 2004; Sliva, 2004). An animated presentation
(www.promega.com
/paguide/animation/selector.htm?coreName=pi3k01) that
shows some events associated with the PI3-K pathway is
available.
In Drosophila, PI3-K is implicated in regulating cell growth
without affecting cell division rates. Studies of wing
imaginal discs show that overexpression of the p110 subunit
of PI3-K results in increased growth. Reducing activity
reduces the size of the wing imaginal disc, producing adult
flies with small wings (Vanhaesebroeck et al. 2001). This
size effect does not appear to be tied to differences in cell
division rates. Similar results have been observed in studies
of PI3-K signaling in mouse heart, where cell growth is
affected but cell division rates are not.
PI3-K signaling is also implicated in progression to S phase
and DNA synthesis in cells. PI3-K activity is tied to the
accumulation of cyclin D in cells and may act at a variety
of levels, transcription, post transcription, and post
translation, to affect cyclin D accumulation. PI3-Ks may
also play roles in relieving inhibition of the cell cycle
(Vanhaesebroeck et al. 2001).
PI3-Ks and Cancer
PI3-Ks are implicated in breast, colon, endometrial, head
and neck, kidney, liver, lymphoma, melanoma, sarcoma
and stomach cancers (Sliva, 2004), making them an
important therapeutic target for human cancer therapy. In
fact, PI3-K mutations found in human cancers have
oncogenic activity (Kang et al. 2005), and PI3-K might
mediate its activity through mTOR (Aoki et al. 2001). Cell
motility is one of many cell functions influenced by PI3-K
signaling. Invasive breast cancer MDA-MB-231 cells, have
higher than normal PI3-K activity. Inhibition of PI3-K by
dominant negative mutations of the PI3-K
regulatory/adaptor subunits or treatment with LY 294002
or wortmannin (PI3-K-specific inhibitors) suppresses
motility of these cells (Sliva, 2004). Studies indicate that
PI3-K may play a role in actin cytoskeleton rearrangements,
perhaps through guanosine nucleotide exchange factors
and GTPase-activating proteins (Vanhaesebroeck et al.
2001).
PI3-Ks can also activate NF-κB through a variety of
mechanisms in different cells. In HepG2 cells, IL-1
stimulates the phosphorylation and activation of NF-κB
through a PI3-K-dependent pathway (Sliva, 2004).
Expression of a dominant negative regulatory subunit of
PI3-K or treatment with PI3-K inhibitors suppressed NF-κB
activation as well as motility in the MDA-MB-231 cell line
(Sliva, 2004).
A viral oncogene that encodes a variant PI3-K was isolated
from a chicken retrovirus. Expression of this oncogene
increases cellular PI, activates Akt and transforms chicken
embryo fibroblasts (Rameh and Cantley, 1999). These
Protocols & Applications Guide
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oncogenic effects may be mediated through the pathways
by which PI3-Ks normally influence cell growth, cell cycle
progression and transcription.
PI3-K signaling is balanced by the activities of inositol lipid
phosphatases. The most well studied PI phosphatase is
PTEN, which was first described as a tumor suppressor
that is deleted or mutated in several human cancers (Rameh
and Cantley, 1999). Furthermore, physical interaction of
PTEN with the MSP58 oncogene inhibits cellular
transformation, thus validating the role of PTEN as a tumor
suppressor (Okamura et al. 2005).
C. Investigating Phosphatases and Kinases as Potential
Therapeutic Targets
The human genome is reported to contain 518 protein
kinases that are involved in phosphorylation of 30% all
cellular proteins (Manning et al. 2002). Taken together,
genes for protein kinases and phosphatases represent five
percent of the human genome (Cohen, 2001). Many other
phosphotransferases play equally important roles in cellular
reactions that use ATP as substrate but are not classified
as protein kinases. These include PI3-kinases (Shears, 2004),
lipid kinases such as sphingosine kinases (French et al. 2003)
and sugar kinases such as glucokinase (Grimsby et al. 2003).
Changes in the level, activity or localization of these kinases,
phosphotransferases and phosphatases greatly influence
the regulation of key cellular processes. Because of the role
that these enzymes play in cellular functions and in various
pathologies, they represent important drug targets (Cohen,
2002). By 2002, more than twenty-six small molecule
inhibitors of protein kinases alone were either approved
for clinical use or in phase I, II or III clinical trials (Cohen,
2002; Pearson and Fabbro, 2004).
This chapter describes the tools available for investigating
the activities of kinases and phosphatases that are involved
in signaling cascades. We describe a variety of technologies
including luminescent and fluorescent assays for kinase
and phosphatases. The phosphorylation state of the
substrates of kinases can also be informative when studying
cell signaling. We describe a variety of antibodies for
detecting the phosphorylated forms of some kinase
substrates as well as kinase substrates and inhibitors that
can be used as tools to analyze kinase activities in samples.
II. Kinase Activity Assays
A. Luminescent Kinase Assays
Kinases are enzymes that catalyze the transfer of a
phosphate group from ATP to a substrate. The depletion
of ATP as a result of kinase activity can be monitored in a
highly sensitive manner through the use of Kinase-Glo®
or Kinase-Glo® Plus Reagent, which uses luciferin, oxygen
and ATP as substrates in a reaction that produces
oxyluciferin and light (Figure 7.2).
The Kinase-Glo® and Kinase-Glo® Plus Reagents rely on
the properties of a proprietary thermostable luciferase
(Ultra-Glo Recombinant Luciferase) that is formulated
to generate a stable “glow-type” luminescent signal. The
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