A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation.
A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.
Ohno
S
S
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Konno
Y
Y
Akita
Y
Y
Yano
A
A
Suzuki
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006706:Homeostasis; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015345:Oligonucleotide Probes; D015240:Phorbol 12,13-Dibutyrate; D000255:Adenosine Triphosphate; D004247:DNA; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate