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Ectoprotein kinase activities on non-differentiated and differentiated U-937 cells
1Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Cellular Signalling
|May 1, 1995
Summary
U-937 cells exhibit ectokinase activity, phosphorylating substrates like phosvitin and protein kinase C (PKC)-peptide. This extracellular phosphorylation is regulated by cell differentiation and may influence monocyte functions.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- U-937 cells are a human monocyte cell line.
- Extracellular enzymes play roles in cell signaling and function.
- Ectokinases are enzymes located on the cell surface.
Purpose of the Study:
- To investigate the presence and characteristics of ectokinase activity on intact U-937 cells.
- To identify substrates and regulatory factors of these cell surface kinases.
Main Methods:
- Incubation of U-937 cells with [gamma-32P] ATP.
- Phosphorylation assays using various peptide substrates (phosvitin, kemptide, PKC-peptide).
- Treatment with kinase inhibitors (Staurosporine, H-7, PKI), cyclic AMP, phorbol ester, and assessment during cell differentiation.
Main Results:
- Rapid ATP incorporation into phosvitin, kemptide, and PKC-peptide was observed.
- Specific inhibitors affected kemptide and PKC-peptide phosphorylation, but not phosvitin.
- Cyclic AMP and phorbol ester modulated kemptide and PKC-peptide phosphorylation.
- Cell differentiation increased phosvitin and PKC-peptide phosphorylation.
- Extracellular phosphorylation occurred at physiologically relevant ATP concentrations.
Conclusions:
- U-937 cells possess at least three distinct ectokinase activities.
- These ectokinases phosphorylate specific substrates on the cell surface.
- Ectokinase activity is modulated by cell differentiation and signaling molecules.
- These findings suggest a role for ectokinases in regulating monocyte-associated functions.