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 agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat 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 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed 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 increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.
Kerr
M
M
Department of Oral Biology, University of Florida, Gainesville 32610.
Fischer
J E
JE
Purushotham
K R
KR
Gao
D
D
Nakagawa
Y
Y
Maeda
N
N
Ghanta
V
V
Hiramoto
R
R
Chegini
N
N
Humphreys-Beher
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002455:Cell Division; D002461:Cell Line, Transformed; D018844:Cyclin-Dependent Kinases; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010306:Parotid Gland / Q000166:cytology / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C447612:CDK11a protein, human; C501852:Cdk11b protein, mouse; D018844:Cyclin-Dependent Kinases; D007545:Isoproterenol