141
5. Remove mouse from imaging instrument and monitor for
complete recovery from anesthesia.
6. Quantify imaging data by region-of-interest (ROI) analysis of
bioluminescence produced by the tumor, using units of photon
flux (Fig. 4a; see Notes 11–17).
The method described herein is an adaptation of the traditional
protein complementation assay for the detection of protein- protein
interaction in live cells. Instead of monitoring the interaction of
two proteins through the use of split reporter molecules, we have
adapted the assay such that the interaction between the “bait” and
the “prey” occurs in response to the activity of a specific kinase.
The kinase can be a serine/threonine- or a tyrosine-kinase. The
reporter has also been engineered such that increased complementation (and therefore reporter activity) occurs in response to
decreased kinase activity. This approach is therefore very well suited
for high-throughput screens for kinase inhibitor libraries since a
positive hit would be detected as an increase in bioluminescence
activity, thereby less likely to result in false positives. We have also
used analogous reporters for whole genome siRNA screens. As an
example, a reporter for TGF-β receptor serine/threonione kinase
activity was used in a human kinome siRNA screen to yield a number of novel genes as regulators of the TGF-β receptor function
[12]. Regulation of the molecular events that lead to the activation
and/or inactivation of the ATM kinase activity is yet to be defined;
therefore, it is anticipated that analogous whole genome siRNA
screens against the ATMR will most likely yield new insights into
the role of novel genes in the regulation of the cellular response to
DNA damage.
4 Notes
1. Substrate sequence for the construction of the reporter should
be decided based on literature searches. It is imperative to have
good antibodies available to detect changes in the substrate
phosphorylation as this will help in validating the kinase
reporter bioluminescence data by biochemical techniques.
2. The selection of a specific cell line for creating a reporterexpressing stable line should be based on the specific questions
being interrogated and the intrinsic activity and detectability of
the kinase and the substrate in the cell line. This should be
determined by experimentation as well as literature searches.
We selected the D54 glioblastoma cell line because ATM inhibition in glioblastoma may sensitize them to IR and chemotherapy [35]. D54 cells express wild-type ATM protein and
responds to ATM inhibitors and irradiation as seen by Western
3.4 Conclusions
Molecular Imaging of ATM Kinase Activity
5. Remove mouse from imaging instrument and monitor for
complete recovery from anesthesia.
6. Quantify imaging data by region-of-interest (ROI) analysis of
bioluminescence produced by the tumor, using units of photon
flux (Fig. 4a; see Notes 11–17).
The method described herein is an adaptation of the traditional
protein complementation assay for the detection of protein- protein
interaction in live cells. Instead of monitoring the interaction of
two proteins through the use of split reporter molecules, we have
adapted the assay such that the interaction between the “bait” and
the “prey” occurs in response to the activity of a specific kinase.
The kinase can be a serine/threonine- or a tyrosine-kinase. The
reporter has also been engineered such that increased complementation (and therefore reporter activity) occurs in response to
decreased kinase activity. This approach is therefore very well suited
for high-throughput screens for kinase inhibitor libraries since a
positive hit would be detected as an increase in bioluminescence
activity, thereby less likely to result in false positives. We have also
used analogous reporters for whole genome siRNA screens. As an
example, a reporter for TGF-β receptor serine/threonione kinase
activity was used in a human kinome siRNA screen to yield a number of novel genes as regulators of the TGF-β receptor function
[12]. Regulation of the molecular events that lead to the activation
and/or inactivation of the ATM kinase activity is yet to be defined;
therefore, it is anticipated that analogous whole genome siRNA
screens against the ATMR will most likely yield new insights into
the role of novel genes in the regulation of the cellular response to
DNA damage.
4 Notes
1. Substrate sequence for the construction of the reporter should
be decided based on literature searches. It is imperative to have
good antibodies available to detect changes in the substrate
phosphorylation as this will help in validating the kinase
reporter bioluminescence data by biochemical techniques.
2. The selection of a specific cell line for creating a reporterexpressing stable line should be based on the specific questions
being interrogated and the intrinsic activity and detectability of
the kinase and the substrate in the cell line. This should be
determined by experimentation as well as literature searches.
We selected the D54 glioblastoma cell line because ATM inhibition in glioblastoma may sensitize them to IR and chemotherapy [35]. D54 cells express wild-type ATM protein and
responds to ATM inhibitors and irradiation as seen by Western
3.4 Conclusions
Molecular Imaging of ATM Kinase Activity