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CD66 family members are associated with tyrosine kinase activity in human neutrophils
K M Skubitz1, K D Campbell, K Ahmed
1Department of Medicine, University of Minnesota Medical School, Minneapolis, USA.
Insights
Stimulating granulocytes increases phosphorylation of CD66a, a transmembrane protein. Associated tyrosine kinases, including Lyn and Hck, may regulate CD66a function and signal transduction in neutrophils.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Granulocyte activation antigens (Ags) like CD66a are upregulated upon stimulation.
- CD66a is a transmembrane protein, distinct from GPI-anchored CD66b and CD66c.
- Previous work showed CD66a phosphorylation in neutrophils, primarily on tyrosine.
Purpose of the Study:
- To investigate the dynamic changes in CD66a phosphorylation after stimulation.
- To identify protein kinase activities associated with CD66 family members.
- To explore the role of associated kinases in CD66a function and signal transduction.
Main Methods:
- Stimulation of human neutrophils with FMLP, PAF, and TPA.
- Analysis of CD66a phosphorylation levels over time.
- Detection and characterization of protein kinase activities associated with CD66a, CD66b, and CD66c.
- Identification of specific tyrosine kinases (Lyn, Hck) involved.
Main Results:
- CD66a phosphorylation rapidly increased post-stimulation, peaking at 1 minute and returning to baseline by 5 minutes.
- Protein kinase activity, predominantly tyrosine kinase activity, was associated with CD66a, CD66b, and CD66c.
- Lyn and Hck were identified as major contributors to the associated tyrosine kinase activity.
Conclusions:
- Tyrosine phosphorylation of CD66a by associated kinases likely plays a role in its function.
- Associated tyrosine kinases may mediate signal transduction from CD66a, CD66b, and CD66c, regulating cellular functions.
Abstract:
The granulocyte activation Ags, CD66a, CD66b, CD66c, and CD66d, are expressed at low levels on resting blood granulocytes, but their surface expression is up-regulated following stimulation. CD66a, in contrast to CD66b and CD66c which are anchored to the membrane via a glycosyl-phosphatidylinositol linkage, is a transmembrane protein with a cytoplasmic domain. We have previously reported that CD66a is phosphorylated in human neutrophils, largely on tyrosine, with a lower level of phosphoserine. We have now found that CD66a undergoes a rapid increase in phosphorylation following stimulation with FMLP, platelet-activating factor, and 12-O-tetradecanoyl-phorbol-13-acetate. This increase in phosphorylation was transient, with maximal phosphorylation observed by 1 min and a return to base line by 5 min following stimulation. Protein kinase activity was detected in neutrophils associated with CD66a, CD66b, and CD66c. Most of the protein kinase activity associated with these Ags was tyrosine kinase activity, with a lesser amount of threonine and serine kinase activities. Lyn and Hck accounted for much of the associated tyrosine kinase activity. The data suggest that phosphorylation of CD66a on tyrosine by an associated tyrosine kinase may play a role in the function of CD66a. In addition, associated tyrosine kinase activity may play a role in signal transduction from CD66a, CD66b, and CD66c to regulate other cell functions.