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Csk suppression of Src involves movement of Csk to sites of Src activity
1Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Abstract:
Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.
Insights
The C-terminal Src kinase (Csk) regulates Src family kinases. Csk
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Csk phosphorylates Src family kinases (SFKs), suppressing their activity.
- Regulation of Csk activity itself is not well understood.
- SFKs play critical roles in cell signaling, proliferation, and migration.
Purpose of the Study:
- To investigate the structural domains of Csk essential for Src suppression.
- To determine how Csk localization is affected by Src activity.
- To elucidate the mechanisms by which Csk regulates SFK activity and localization.
Main Methods:
- Expression of wild-type and mutant Csk in a csk-deficient cell line.
- In vitro kinase assays to assess Csk catalytic activity.
- Immunofluorescence microscopy to analyze the subcellular localization of Csk and Src.
Main Results:
- Wild-type Csk suppressed Src activity, while mutants in SH2, SH3, and catalytic domains did not.
- SH2 domain mutants of Csk were catalytically inactive, whereas an SH3 deletion mutant retained activity.
- Csk relocalized to podosomes upon Src activation, and this localization required functional SH2 and SH3 domains.
- A catalytically inactive Csk mutant localized with active Src to podosomes, indicating catalytic activity is not required for localization.
Conclusions:
- Both SH2 and SH3 domains of Csk are crucial for its ability to suppress Src activity and for its proper localization to sites of active Src.
- Csk localization to podosomes is dependent on active Src but not solely on Csk binding to phosphorylated Src.
- These findings highlight the importance of Csk's structural domains in mediating its regulatory function and subcellular targeting.
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