Differential regulation of adapter proteins Dok2 and Dok1 in platelets, leading to an association of Dok2 with integrin alphaIIbbeta3.
BACKGROUND
We previously demonstrated that Dok2 is rapidly phosphorylated on tyrosine residues in platelets in response to thrombin, the immunoreceptor tyrosine-based activation motif-coupled collagen receptor glycoprotein (GP) VI, and by integrin alphaIIbbeta3.


OBJECTIVE
In this study we further delineate the regulation of phosphorylation of Dok2 and compare this to the related adapter Dok1.


RESULTS
We demonstrate expression of Dok1 in platelets and the unexpected observation that the adapter protein undergoes tyrosine phosphorylation in response to thrombin but not to GPVI or integrin alphaIIbbeta3. Furthermore, Dok1 phosphorylation is transient, peaking at 30 s and returning to basal by 5 min, whereas Dok2 phosphorylation is delayed but sustained. Dok2 phosphorylation, but not that of Dok1, is inhibited by Src kinase inhibitors and by chelation of intracellular calcium. Further, phosphorylation of Dok2 by thrombin and integrin alphaIIbbeta3 in mouse platelets is independent of Syk and phospholipase Cgamma2. Additionally, Dok2 coimmunoprecipitates with integrin alphaIIbbeta3 downstream of Src kinases.


CONCLUSIONS
These results demonstrate differential modes of regulation of Dok1 and Dok2 in platelets. Further, they raise the interesting possibility that Dok2 plays an important role in integrin outside-in signaling through a physical and functional interaction with integrin alphaIIbbeta3.
Hughan
S C
SC
Centre for Cardiovascular Sciences, Institute of Biomedical Research, University of Birmingham, Birmingham, UK. sascha.hughan@med.monash.edu.au
Watson
S P
SP
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C115089:DOK2 protein, human; C489561:Dok1 protein, mouse; C498444:Dok2 protein, mouse; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D016601:RNA-Binding Proteins