Format

Send to

Choose Destination
Methods Mol Biol. 2016;1360:157-67. doi: 10.1007/978-1-4939-3073-9_12.

Characterization of an Engineered Src Kinase to Study Src Signaling and Biology.

Author information

1
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
2
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, 60612, USA.
3
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. cjder@med.unc.edu.

Abstract

Pharmacologic inhibitors of protein kinases comprise the vast majority of approved signal transduction inhibitors for cancer treatment. An important facet of their clinical development is the identification of the key substrates critical for their driver role in cancer. One approach for substrate identification involves evaluating the phosphorylation events associated with stable expression of an activated protein kinase. Another involves genetic or pharmacologic inhibition of protein kinase expression or activity. However, both approaches are limited by the dynamic nature of signaling, complicating whether phosphorylation changes are primary or secondary activities of kinase function. We have developed rapamycin-regulated (RapR) protein kinases as molecular tools that allow for the study of spatiotemporal regulation of signaling. Here we describe the application of this technology to the Src tyrosine kinase and oncoprotein (RapR-Src). We describe how to achieve stable expression of this tool in cell lines and how to subsequently activate the tool and determine its function in signaling and morphology.

KEYWORDS:

FK506-binding protein; KRAS; Oncogene; Pancreatic cancer; Rapamycin; Src; Tyrosine kinase; mTOR

PMID:
26501909
PMCID:
PMC4621786
DOI:
10.1007/978-1-4939-3073-9_12
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Springer Icon for PubMed Central
Loading ...
Support Center