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c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein
1Department of Biochemistry, School of Medicine, Temple University, Philadelphia, Pennsylvania 19140, USA.
Abstract:
The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase. Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.
Insights
A novel protein, ABL-associated protein 1 (AAP1), inhibits c-ABL tyrosine kinase activity. This regulation is lost in oncogenic forms like BCR-ABL, suggesting AAP1 acts as a trans regulator of c-ABL kinase.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- The c-ABL tyrosine kinase's activity is negatively regulated by its Src homology domain 3 (SH3).
- Loss or mutation of the SH3 domain activates c-ABL, leading to cellular transformation.
- This regulation is hypothesized to involve cellular proteins interacting with the SH3 domain.
Purpose of the Study:
- To identify proteins that interact with the c-ABL SH3 domain.
- To characterize the function of interacting proteins in c-ABL kinase regulation.
- To investigate the role of these interactions in the oncogenicity of c-ABL variants.
Main Methods:
- Yeast two-hybrid screening using c-ABL SH3SH2 domains as bait.
- Identification of interacting proteins via cDNA library screening.
- Kinase assays to assess the effect of identified proteins on c-ABL activity.
Main Results:
- A novel protein, ABL-associated protein 1 (AAP1), was identified that binds to c-ABL SH3SH2 domains.
- AAP1 failed to bind to the SH3 domain of the BCR-ABL oncoprotein.
- AAP1 inhibited the kinase activity of c-ABL but had minimal effect on v-ABL and BCR-ABL.
- Loss of susceptibility to AAP1 regulation correlated with the oncogenicity of activated c-ABL forms.
Conclusions:
- AAP1 functions as a trans regulator, inhibiting c-ABL tyrosine kinase activity.
- The inability of AAP1 to regulate oncogenic ABL variants suggests a mechanism for their activation.
- These findings provide insights into the regulation of c-ABL and its role in cancer.