Analysis of the subcellular distribution of protein kinase Calpha using PKC-GFP fusion proteins.
One important factor for the determination of the specific functions of protein kinase C (PKC) isoforms is their specific subcellular localization. In NIH 3T3 fibroblasts phorbol esters induce translocation of PKCalpha to the plasma membrane and the nucleus. In order to investigate PKCalpha's subcellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKCalpha and the green fluorescent protein (GFP). Purified GFP-PKCalpha from baculovirus-infected insect cells undergoes nuclear accumulation without any further stimuli in digitonin-permeabilized cells. Interestingly, permeabilization appears to be a trigger for PKCalpha's nuclear translocation, since the fusion protein also translocates to the nucleus in transiently transfected cells following permeabilization. This suggests that PKCalpha has a high nuclear binding capacity even in the case of large protein amounts. In contrast to endogenous PKCalpha, overexpressed GFP-PKCalpha as well as overexpressed PKCalpha itself translocates mainly to the plasma membrane and only to a smaller extent to the nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKCalpha enabled determination of motifs involved PKCalpha's subcellular distribution: A25E and K368R point mutations of PKCalpha showed enhanced affinity for the plasma membrane, whereas sequences within the regulatory domain probably confer PKCalpha's nuclear accumulation.
Wagner
S
S
Freie Universität Berlin, Institut für Chemie, Biochemie, Germany.
Harteneck
C
C
Hucho
F
F
Buchner
K
K
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011493:Protein Kinase C / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D018411:Spodoptera; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha