Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase Calpha in response to diacylglycerol.
We have seen that protein kinase Calpha (PKCalpha) is transiently translocated to the plasma membrane by carbachol stimulation of neuroblastoma cells. This is induced by the Ca2+ increase, and PKCalpha does not respond to diacylglycerol (DAG). The unresponsiveness is dependent on structures in the catalytic domain of PKCalpha. This study was designed to investigate if and how the kinase activity and autophosphorylation are involved in regulating the translocation. PKCalpha enhanced green fluorescent protein translocation was studied in living neuroblastoma cells by confocal microscopy. Carbachol stimulation induced a transient translocation of PKCalpha to the plasma membrane and a sustained translocation of kinase-dead PKCalpha. In cells treated with the PKC inhibitor GF109203X, wild-type PKCalpha also showed a sustained translocation. The same effects were seen with PKCbetaI, PKCbetaII, and PKCdelta. Only kinase-dead and not wild-type PKCalpha translocated in response to 1,2-dioctanoylglycerol. To examine whether autophosphorylation regulates relocation to the cytosol, the autophosphorylation sites in PKCalpha were mutated to glutamate, to mimic phosphorylation, or alanine, to mimic the non-phosphorylated protein. After stimulation with carbachol, glutamate mutants behaved like wild-type PKCalpha, whereas alanine mutants behaved like kinase-dead PKCalpha. When the alanine mutants were treated with 1,2-dioctanoylglycerol, all cells showed a sustained translocation of the protein. However, neither carbachol nor GF109203X had any major effects on the level of autophosphorylation, and GF109203X potentiated the translocation of the glutamate mutants. We, therefore, hypothesize that 1) autophosphorylation of PKCalpha limits its sensitivity to DAG and 2) that kinase inhibitors augment the DAG sensitivity of PKCalpha, perhaps by destabilizing the closed conformation.
Stensman
Helena
H
Lund University, Molecular Medicine, Entrance 78, 3rd Floor, Malmö University Hospital, 205 02 Malmö, Sweden.
Raghunath
Arathi
A
Larsson
Christer
C
D000409:Alanine / Q000737:chemistry; D015153:Blotting, Western; D002217:Carbachol / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D018613:Microscopy, Confocal; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport; D013997:Time Factors; D014162:Transfection
D018679:Cholinergic Agonists; D004075:Diglycerides; D007211:Indoles; D008301:Maleimides; D002217:Carbachol; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C070515:bisindolylmaleimide I; D000409:Alanine