Abstract
In the present study, we assessed the cooperative roles of C-terminal Src kinase (Csk) binding protein (Cbp) and Caveolin-1 (Cav-1) in the mechanism of Src family tyrosine kinase (SFK) inhibition by Csk. SFKs are inactivated by phosphorylation of their C-terminal tyrosine by Csk. Whereas SFKs are membrane-associated, Csk is a cytoplasmic protein and therefore requires membrane adaptors such as Cbp or Cav-1 for recruitment to the plasma membrane to mediate SFK inhibition. To determine the specific role of Cav-1 and Cbp in SFK inhibition, we measured c-Src activity in the absence of each membrane adaptor. It is noteworthy that in lungs and fibroblasts from Cav-1(−/−) mice, we observed increased expression of Cbp compared with wild-type (WT) controls. However, both c-Src activity and Csk localization at the membrane were similar between Cav-1(−/−) fibroblasts and WT cells. Likewise, Cbp depletion by small interfering RNA (siRNA) treatment of WT cells had no effect on basal c-Src activity, but it increased the phosphorylation state of Cav-1. Immunoprecipitation then confirmed increased association of Csk with phosphomimicking Cav-1. Knockdown of Cbp by siRNA in Cav-1(−/−) cells revealed increased basal c-Src activity, and re-expression of WT Cav-1 in the same cells reduced basal c-Src activity. Taken together, these results indicate that Cav-1 and Cbp cooperatively regulate c-Src activity by recruiting Csk to the membrane where it phosphorylates c-Src inhibitory tyrosine 529. Furthermore, when either Cav-1 or Cbp expression is reduced or absent, there is a compensatory increase in the phosphorylation state or expression level of the other membrane-associated Csk adaptor to maintain SFK inhibition.
Footnotes
This research was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL71626, P01-HL60678]; and the American Heart Association Midwest Affiliate [Grant 0910026G].
This article is based on a thesis submitted in partial fulfillment of the requirements for a doctoral degree: Place AT (2011) Molecular mechanism of Src regulation by Csk, SHP-2, Cbp, and caveolin-1. Ph.D. thesis, Department of Pharmacology, University of Illinois, Chicago, IL.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.073957.
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ABBREVIATIONS:
- SFK
- Src family kinase
- Csk
- C-terminal Src kinase
- Cbp
- Csk-binding protein
- MF
- mouse fibroblasts
- SH
- Src homology
- WT
- wild type
- siRNA
- small interfering RNA
- PAGE
- polyacrylamide gel electrophoresis
- RIPA
- radioimmunoprecipitation assay
- PMSF
- phenylmethylsulfonyl fluoride
- ODG
- n-octylglucoside
- TBST
- Tris-buffered saline/Tween 20
- HBSS+/+
- Hanks' balanced salt solution with Ca2+ and Mg2+
- ELISA
- enzyme-linked immunosorbent assay
- HEK
- human embryonic kidney
- GFP
- green fluorescent protein
- S
- scrambled.
- Received June 3, 2011.
- Accepted July 21, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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