Characterization of an Engineered Src Kinase to Study Src Signaling and Biology.
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.
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
Leanna R
LR
Gentry
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, 60612, USA.
Andrei V
AV
Karginov
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
Klaus M
KM
Hahn
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. cjder@med.unc.edu.
Channing J
CJ
Der
D002461:Cell Line, Transformed; D005813:Genes, Synthetic; D011905:Genes, ras; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D010766:Phosphorylation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020123:Sirolimus / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000235:genetics; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics; D019061:src-Family Kinases / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D019061:src-Family Kinases; D000097573:Red Fluorescent Protein; C546842:MTOR protein, human
FK506-binding protein; KRAS; Oncogene; Pancreatic cancer; Rapamycin; Src; Tyrosine kinase; mTOR