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Dok-R binds c-Abl and regulates Abl kinase activity and mediates cytoskeletal reorganization
Zubin Master1, Jennifer Tran, Aseem Bishnoi
1Division of Molecular and Cellular Biology, Sunnybrook and Women's College Health Sciences Centre, Toronto M4N 3M5, Canada.
Abstract:
Dok-R, also known as Dok-2/FRIP, belongs to the DOK family of signaling molecules that become tyrosine-phosphorylated by several different receptor and cytoplasmic tyrosine kinases. Tyrosine phosphorylation of DOK proteins establishes high affinity binding sites for other signaling molecules leading to activation of a signaling cascade. Here we show that Dok-R associates with c-Abl directly via a constitutive SH3-mediated interaction and that this binding requires a PMMP motif in the proline-rich tail of Dok-R. The Dok-R-Abl interaction is further enhanced by an active c-Abl kinase, which requires the presence of its SH2 domain. Interaction of Dok-R with c-Abl also results in an increase in c-Abl tyrosine phosphorylation and kinase activity. Furthermore, we demonstrate that this increase in kinase activity correlates with a concomitant increase in c-Abl-mediated biological activity as measured by the formation of actin microspikes. Our data are the first to demonstrate that Dok-R and c-Abl interact in both a constitutive and inducible fashion and that Dok-R influences the intracellular kinase and biological activity of c-Abl.
Insights
Dok-R directly binds to c-Abl, enhancing its kinase activity and biological functions. This interaction, crucial for signaling cascades, occurs constitutively and inducibly, highlighting Dok-R
Area of Science:
- Cellular signaling
- Molecular biology
- Protein interactions
Background:
- Dok-R (also known as Dok-2/FRIP) is a signaling molecule in the DOK family.
- DOK proteins are tyrosine-phosphorylated, creating binding sites for downstream signaling molecules.
- This phosphorylation initiates signaling cascades.
Purpose of the Study:
- To investigate the interaction between Dok-R and c-Abl.
- To determine the functional consequences of this interaction on c-Abl activity.
- To elucidate the molecular mechanisms underlying the Dok-R-c-Abl association.
Main Methods:
- Co-immunoprecipitation assays to confirm protein-protein interactions.
- Site-directed mutagenesis to identify critical binding motifs (PMMP motif).
- Kinase assays to measure c-Abl activity.
- Assessment of actin microspike formation as a measure of biological activity.
Main Results:
- Dok-R directly binds to c-Abl through a constitutive SH3-mediated interaction requiring a PMMP motif in Dok-R.
- An active c-Abl kinase domain, specifically its SH2 domain, enhances this interaction.
- Dok-R binding increases c-Abl tyrosine phosphorylation and kinase activity.
- Increased c-Abl activity correlates with enhanced formation of actin microspikes.
Conclusions:
- Dok-R and c-Abl interact both constitutively and inducibly.
- Dok-R positively influences the intracellular kinase and biological activity of c-Abl.
- This interaction represents a novel regulatory mechanism for c-Abl function in cellular signaling.