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CD45 regulation of tyrosine phosphorylation and enzyme activity of src family kinases
C M Burns1, K Sakaguchi, E Appella
1Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Previous analyses have suggested that the CD45 tyrosine phosphatase activates src family tyrosine kinases p56lck and p59fyn by dephosphorylating regulatory COOH-terminal residues. We have examined the status of p56lck and p59fyn in murine and human CD45- T cell lines. Surprisingly, despite the fact that p56lck and p59fyn were spontaneously hyperphosphorylated, the tyrosine kinase activity of both enzymes was increased in CD45- versus CD45+ cells. In vitro exposure of hyperphosphorylated p56lck to CD45 decreased enzyme activity to near-basal levels. Lck from CD45- cells was hyperphosphorylated on the cyanogen bromide digestion fragment that contains the negative regulatory residue Tyr-505, and the identity of this site of phosphorylation was confirmed by trypsin digestion followed by high performance liquid chromatography. Loss of CD45 results, therefore, in a paradoxical hyperphosphorylation of the COOH-terminal tyrosine and increased src family kinase enzymatic activity.
Insights
Loss of CD45 phosphatase paradoxically increases src family kinase activity in T cells. Despite hyperphosphorylation, p56lck and p59fyn show enhanced enzymatic function in CD45- cells.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- CD45 tyrosine phosphatase is known to activate src family tyrosine kinases (SFKs) like p56lck and p59fyn.
- Activation is thought to occur via dephosphorylation of regulatory COOH-terminal residues.
Purpose of the Study:
- To investigate the phosphorylation status and enzymatic activity of p56lck and p59fyn in CD45- T cells.
- To elucidate the role of CD45 in regulating SFK activity.
Main Methods:
- Analysis of p56lck and p59fyn in murine and human CD45- T cell lines.
- In vitro kinase assays.
- Cyanogen bromide and trypsin digestion followed by high-performance liquid chromatography (HPLC) to identify phosphorylation sites.
Main Results:
- p56lck and p59fyn were found to be hyperphosphorylated in CD45- T cells.
- Despite hyperphosphorylation, the tyrosine kinase activity of p56lck and p59fyn was increased in CD45- cells compared to CD45+ cells.
- In vitro, CD45 reduced the activity of hyperphosphorylated p56lck.
- Hyperphosphorylation occurred at the negative regulatory residue Tyr-505.
Conclusions:
- Loss of CD45 leads to paradoxical hyperphosphorylation of the COOH-terminal tyrosine residue (Tyr-505) in src family kinases.
- This hyperphosphorylation is associated with increased, not decreased, src family kinase enzymatic activity in CD45- T cells.