TSLA23-NRK CELLS NEED PP60V-SRC PROTEIN-TYROSINE KINASE-ACTIVITY IN G2 PHASE TO INITIATE MITOSIS IN SERUM-FREE MEDIUM

Lowering the temperature from 41 to 36-degrees-C stimulates quiescent tsLA23-NRK rat cells (infected with the tsLA23 mutant of the Rous sarcoma virus) in serum-free medium to resume cycling and initiate DNA replication by reactivating the tsLA23-RSV's abnormally thermolabile pp60v-src protein-tyrosine kinase. Inactivating the enzyme in these pp60v-src-stimulated cells by again raising the temperature to 41-degrees-C after the cells had initiated DNA replication did not prevent the completion of DNA replication and entry into the G2 phase, but it stopped the initiation of mitosis. Adding serum at the time of the temperature increase replaced the lost pp60v-src activity and the cells were able to continue to mitosis. The G2-arrested cells at 41-degrees-C were able to intiate mitosis when pp60v-src was reactivated again by lowering the temperature to 36-degrees-C. These observations suggest that protein-tyrosine kinase activity is needed to initiate mitosis and that the tsLA23-NRK cell is a good model for studying the function of this kinase activity in the initiation of mitosis.