Overexpression of the type II regulatory subunit of the cAMP-dependent protein kinase eliminates the type I holoenzyme in mouse cells.
Mammalian tissues and cell lines express two major types of cAMP-dependent protein kinase, PKA-I and PKA-II, which can be distinguished at the molecular level by the presence of either type I or type II regulatory subunits in the holoenzyme. An expression vector for the mouse type II regulatory subunit (RII alpha) was transfected into ras-transformed NIH3T3 (R3T3) cells, which contain approximately equal amounts of both holoenzymes, PKA-I and PKA-II. In RII alpha-overexpressing R3T3 cells, PKA-II levels were increased, and the level of PKA-I declined. The decrease in PKA-I was dependent on the amount of RII alpha expressed, and at high levels of RII alpha expression, PKA-I was completely eliminated. In contrast, overexpression of the type I regulatory subunit (RI alpha) did not alter PKA isozyme levels. We propose that competition between RII alpha and RI alpha for a limited pool of catalytic subunit results in preferential assembly of PKA-II and that significant amounts of PKA-I are formed only if catalytic subunit is present in excess of the RII alpha subunit. The PKA-I isozyme, which is absent in untransformed 3T3 cells, is not essential for the transformed phenotype of R3T3 cells. RII alpha-overexpressing R3T3 cells that are devoid of PKA-I continued to exhibit a transformed phenotype including anchorage-independent growth. Overexpression of RII alpha provides a genetic approach that may prove useful in demonstrating specific functions for the two PKA isozymes in cAMP-dependent signal transduction pathways.
Otten
A D
AD
Department of Pharmacology, University of Washington, Seattle 98195.
McKnight
G S
GS
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006706:Homeostasis; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D007527:Isoenzymes; D046911:Macromolecular Substances; D011494:Protein Kinases