CHARACTERIZATION OF THE SYNTHESIS AND EXPRESSION OF THE GTA-KINASE FROM TRANSFORMED AND NORMAL RODENT CELLS

The murine transformed cell line YC-8 and beta-adrenergic receptor ago nist (isoproternol) treated rat and mouse parotid gland acinar cells e ctopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression o f the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones fo r the protein coding region of the kinase were isolated by reverse tra nscriptase - PCR cloning. DNA sequence analysis failed to show sequenc e differences with the normal homolog from mouse cells although Southe rn blot analysis of YC-8, and a second cell line K181, indicated chang es in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The ra t cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 an d a control cell line LSTRA revealed the synthesis of a major 3.0 kb m RNA from both cell lines plus the unique expression of a 4.5 kb mRNA i n the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirme d the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increas ed rate of DNA synthesis, cell number and ectopic expression of cell s urface galactosyltransferase in the sense orientation. Antisense expre ssion or vector alone did not alter growth characteristics of acinar c ells. A polyclonal antibody monospecific to a murine amino terminal pe ptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. Ho wever, upon growth stimulation, kinase was detected primarily in a pen inuclear and nuclear immunostaining pattern. Western blot analysis con firmed a translocation from a cytoplasmic localization in both LSTRA a nd quiescent salivary cells to a membrane-associated localization in Y C-8 and proliferating salivary cells.