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Phosphorylation of the multidrug resistance associated glycoprotein
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Abstract:
Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.
Insights
Multidrug resistance involves a glycoprotein whose phosphorylation is regulated by cyclic AMP (cAMP) and protein kinase A. This suggests a key role for protein kinase A in maintaining drug resistance in cancer cells.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) is a significant challenge in cancer therapy.
- The MDR phenotype is often associated with the overexpression of membrane glycoproteins, such as P-glycoprotein.
- Understanding the regulation of these glycoproteins is crucial for developing strategies to overcome drug resistance.
Purpose of the Study:
- To investigate the phosphorylation of the P-glycoprotein in drug-resistant cells.
- To identify the kinases involved in P-glycoprotein phosphorylation.
- To explore the relationship between P-glycoprotein phosphorylation and the multidrug resistance phenotype.
Main Methods:
- Studied phosphorylation of P-glycoprotein in intact cells and cell-free membrane fractions.
- Used immunoprecipitation with a specific antibody to detect phosphorylated P-glycoprotein.
- Performed phosphorylation assays with partially purified membrane fractions and cAMP-dependent protein kinase A.
Main Results:
- P-glycoprotein was identified as a phosphoprotein.
- Phosphorylation of P-glycoprotein was enhanced by cyclic AMP (cAMP).
- The catalytic subunit of cAMP-dependent protein kinase A phosphorylated P-glycoprotein, indicating its involvement.
Conclusions:
- P-glycoprotein is a phosphoprotein regulated by cAMP and protein kinase A.
- Membrane-associated protein kinase A is likely involved in P-glycoprotein phosphorylation.
- The phosphorylation state of P-glycoprotein may be critical for maintaining the multidrug resistance phenotype.