Protein kinase C and control of the A system amino acid transporter

Author

Michael Henry Bast

Date of Award

12-1990

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

John S. Cook

Committee Members

Steven J. Kennel, Fred Snyder, Margaret Terzaghi-Howe

Abstract

The activity of the A System amino acid transporter correlates with the growth state of the cell. Rapidly growing LLC-PK₁ cells have a high rate of A System transport activity, and confluent, differentiated LLC-PK₁ cells have a low rate of A System transport activity. Differentiated LLC-PK₁ cells treated with tumor promoters such as TPA will increase A System transport activity. Addition of the diphenolic compounds quercetin, phloretin and diethylstilbestrol to LLC-PK₁ cells will prevent the TPA stimulation of A System transport. This is shown to be due to inhibition of protein kinase C, part of a major signal transduction pathway, and not to an interaction with the transporter. Active protein kinase C is only required for approximately 10 % of the time needed to generate a TPA response, indicating a possible cascade reaction is involved in TPA stimulation. Changes in transport were shown to be due to changes in the V_max of transport, indicating that transport is controlled by changing the number of active transporters, rather that individual transporter activity. This was shown for transport changes due to growth state, TPA stimulation of differentiated LLC-PK₁ cells, or inhibition of protein kinase C in rapidly growing cells by quercetin. Polyclonal antisera were raised against the regulatory and catalytic domains of protein kinase C to determine if the calpain-generated kinase fragment, PKM, has a role in stimulation of A System transport activity. Although the antibodies react with protein kinase C in solution, and will react on immunoblots with protein kinase C from pig or rat brain, the antisera do not react with an appropriate molecular weight band in LLC-PK₁ cells. Instead, bands were seen at positions similar to those reported for a newly discovered protein kinase C isotype, PKC ς.

Recommended Citation

Bast, Michael Henry, "Protein kinase C and control of the A system amino acid transporter. " PhD diss., University of Tennessee, 1990.
https://trace.tennessee.edu/utk_graddiss/11268