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DOI: 10.1055/s-2002-33259
Effects of the Putatively Oncogenic Protein Kinase Cα D294G Mutation on Enzymatic Activity and Cell Growth and its Occurrence in Human Thyroid Neoplasias
Publikationsverlauf
Received 30 July 2001
Accepted after Revision 20 February 2002
Publikationsdatum:
12. August 2002 (online)
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Abstract
A point mutation of protein kinase Cα (PKCα) has been described in pituitary adenomas and in follicular adenomas and thyroid carcinomas. The mutation results in an exchange of aspartic acid into a glycine of the amino acid 294 of PKCα, which is located adjacent to the Ca2+-binding hinge region and has been proposed as an activation inhibitor. To investigate its biochemical sequelae, we constructed the mutated enzyme and expressed it in human embryonic kidney cells (HEK). The KM of the purified enzyme for Ca2+ and its KM for the substrate MBP4 - 14 was not altered by the mutation. Translocation of PKCα to HEK cell membranes upon activation was not changed and the mutant potently inhibited cell proliferation upon 5-fold stable overexpression in HEK cells. Thus, loss of function in mutated PKCα was excluded. A screen for the mutation using a restriction assay with a sensitivity of at least 8 % for the mutated DNA did not show any mutation in 11 carcinoma and 13 adenomatous thyroid samples. We conclude that the A294G mutation of PKCα does not detectably affect its biochemical properties in vitro or in vivo, and is at least rare in thyroid neoplasias, in Germany.
Keywords
Purification - Enzyme Activity - Mutagenesis - Cell Growth - HEK Cells
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