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Identification of acidic amino acid residues in the protein kinase C alpha V5 domain that contribute to its insensitivity to diacylglycerol

Stensman, Helena LU and Larsson, Christer LU (2007) In Journal of Biological Chemistry 282(39). p.28627-28638
Abstract
The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with... (More)
The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC alpha indicating a conformational difference elicited by the mutations. Expression of the isolated PKC alpha V5 domain led to increased diacylglycerol sensitivity of PKC alpha. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC alpha sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC alpha that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC alpha in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC alpha. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
282
issue
39
pages
28627 - 28638
publisher
ASBMB
external identifiers
  • wos:000249642100037
  • scopus:35349021416
ISSN
1083-351X
DOI
10.1074/jbc.M702248200
language
English
LU publication?
yes
id
be989558-79e8-4085-ac33-f157bce991b7 (old id 656587)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=17673466&dopt=Abstract
date added to LUP
2007-12-20 12:04:12
date last changed
2017-03-19 03:33:50
@article{be989558-79e8-4085-ac33-f157bce991b7,
  abstract     = {The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC alpha indicating a conformational difference elicited by the mutations. Expression of the isolated PKC alpha V5 domain led to increased diacylglycerol sensitivity of PKC alpha. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC alpha sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC alpha that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC alpha in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC alpha.},
  author       = {Stensman, Helena and Larsson, Christer},
  issn         = {1083-351X},
  language     = {eng},
  number       = {39},
  pages        = {28627--28638},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Identification of acidic amino acid residues in the protein kinase C alpha V5 domain that contribute to its insensitivity to diacylglycerol},
  url          = {http://dx.doi.org/10.1074/jbc.M702248200},
  volume       = {282},
  year         = {2007},
}