Phorbol ester activation of functional rat protein kinase C beta-1 causes phenotype in yeast.
The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. The molecular dissection of its mechanisms of action has been limited in part by the genetic inaccessibility and complexity of signaling in mammalian cells. Here we present a novel approach to study rat PKC beta-1 action in yeast, a simple lower eukaryotic genetic model. Expression of its cDNA in Saccharomyces cerevisiae introduces novel phorbol ester binding sites which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro consistent with a fully functional protein which phosphorylates cellular yeast proteins in vivo. Phorbol ester activation of PKC beta-1 in vivo results in biological responses which include stimulation of extracellular calcium uptake, changes in cell morphology, and an increase in the cell doubling time. These PKC functions are not affected by truncation of 12 amino terminal amino acids; however, they are completely abolished by truncation of 15 or more carboxyl terminal amino acids which likely result in inactivation of the kinase. The increase in the yeast doubling time caused by PKC beta-1 activation provides a phenotype which can be exploited as a screen for the activity of random PKC cDNA mutations. Our findings indicate that rat PKC beta-1 is functional in yeast and leads to biological responses which suggest compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.
Riedel
H
H
Section on Molecular Biology, Joslin Diabetes Center, Boston, Massachusetts 02215.
Hansen
H
H
Parissenti
A M
AM
Su
L
L
Shieh
H L
HL
Zhu
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D007527:Isoenzymes; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C