Characterization of a 170 kD Membrane Protein Kinase which has Increased Activity in KB-V1 Multidrug Resistant Cells
The phenomenon of multidrug resistance, in which tumor cells mutate and become resistant to a series of hydrophobic drugs derived from natural products, creates a major obstacle to effective chemotherapy techniques. Juliano and Ling (1976) discovered an energy-dependent, membrane-bound efflux pump, now called the P-glycoprotein pump (MW = 170 kD), which is overexpressed in many multidrug resistant cell lines and is believed to be responsible for the removal of cytotoxic agents from the cell. Following the discovery that membrane kinases such as PKC and PK-A seem to be related to the regulation of P-glycoprotein, Sampson and Abraham (1992) have found a 170 kD kinase in the cell membrane of the human carcinoma multidrug resistant cell line (KB-V1) that is absent in the corresponding sensitive cell line (KB-3-1). This finding suggests that PK170 is also involved in the multidrug resistance mechanism. The study presented here used the methods of Ferrell and Martin (1989, 1990) and Sampson and Abraham (in preparation) in which the membrane fraction of KB-V-1 cells was isolated, electrophoresed, and exposed to radiolabeled ATP. In this way, autophosphorylation and therefore activity of kinases in the membrane were determined. Results suggest that PK170 is activated by certain serine-threonine kinase inhibitors in the in vitro assay, suggesting that the inhibitors are inhibiting the autophosphorylation of PK170 in the intact cell. Tyrosine kinase inhibitors had no effect on PK170 in this assay, suggesting that PK170 is a serine/threonine protein kinase. The activation of PK170 by K-252b, an impermeable kinase inhibitor, suggests that PK170 may be a cell surface receptor. The attempt to characterize PK170 by the use of interleukins and other various ligands in this assay proved unsuccessful. Further experimentation will be necessary to characterize PK170, to investigate its connection to the multidrug resistance mechanism, and to develop any novel chemotherapy techniques which may result from these findings.