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Autophosphorylation suppresses, whereas kinase inhibition augments, the translocation of PKCa in response to diacylglycerol.

Stensman, Helena LU ; Raghunath, Arathi and Larsson, Christer LU (2004) In Journal of Biological Chemistry 279(39). p.40576-40583
Abstract
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... (More)
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 nonphosphorylated 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. (Less)
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type
Contribution to journal
publication status
published
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in
Journal of Biological Chemistry
volume
279
issue
39
pages
40576 - 40583
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • wos:000223916800045
  • scopus:4644326204
ISSN
1083-351X
DOI
10.1074/jbc.M405560200
language
English
LU publication?
yes
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The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Tumour Cell Biology (013017530)
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ac2cb205-269f-4d6a-885a-b78a8b632c68 (old id 125638)
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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15277524&dopt=Abstract
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2022-01-26 17:17:44
@article{ac2cb205-269f-4d6a-885a-b78a8b632c68,
  abstract     = {{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 nonphosphorylated 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.}},
  author       = {{Stensman, Helena and Raghunath, Arathi and Larsson, Christer}},
  issn         = {{1083-351X}},
  language     = {{eng}},
  number       = {{39}},
  pages        = {{40576--40583}},
  publisher    = {{American Society for Biochemistry and Molecular Biology}},
  series       = {{Journal of Biological Chemistry}},
  title        = {{Autophosphorylation suppresses, whereas kinase inhibition augments, the translocation of PKCa in response to diacylglycerol.}},
  url          = {{http://dx.doi.org/10.1074/jbc.M405560200}},
  doi          = {{10.1074/jbc.M405560200}},
  volume       = {{279}},
  year         = {{2004}},
}