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Proc Natl Acad Sci U S A. 2014 Aug 26;111(34):12420-5. doi: 10.1073/pnas.1404487111. Epub 2014 Aug 12.

Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms.

Author information

1
Department of Pharmacology.
2
Department of Biomedical Engineering.
3
Department of Pharmacology, Department of Biochemistry and Biophysics, and.
4
Department of Pharmacology, khahn@med.unc.edu karginov@uic.edu.
5
Department of Pharmacology, Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 khahn@med.unc.edu karginov@uic.edu.

Abstract

The Src kinase family comprises nine homologous members whose distinct expression patterns and cellular distributions indicate that they have unique roles. These roles have not been determined because genetic manipulation has not produced clearly distinct phenotypes, and the kinases' homology complicates generation of specific inhibitors. Through insertion of a modified FK506 binding protein (insertable FKBP12, iFKBP) into the protein kinase isoforms Fyn, Src, Lyn, and Yes, we engineered kinase analogs that can be activated within minutes in living cells (RapR analogs). Combining our RapR analogs with computational tools for quantifying and characterizing cellular dynamics, we demonstrate that Src family isoforms produce very different phenotypes, encompassing cell spreading, polarized motility, and production of long, thin cell extensions. Activation of Src and Fyn led to patterns of kinase translocation that correlated with morphological changes in temporally distinct stages. Phenotypes were dependent on N-terminal acylation, not on Src homology 3 (SH3) and Src homology 2 (SH2) domains, and correlated with movement between a perinuclear compartment, adhesions, and the plasma membrane.

KEYWORDS:

image analysis; live cell imaging; motion classification; protein engineering; rapamycin

PMID:
25118278
PMCID:
PMC4151743
DOI:
10.1073/pnas.1404487111
[Indexed for MEDLINE]
Free PMC Article
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