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Protein kinase Czeta is a negative regulator of protein kinase B activity
R P Doornbos1, M Theelen, P C van der Hoeven
1Institute of Biomembranes, Department of Molecular Cell Biology, Utrecht University, 3584 CH Utrecht, The Netherlands.
Abstract:
Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.
Insights
Protein kinase B (PKB) interacts with PKCzeta, inhibiting its activity. This interaction, mediated by PKB's AH domain, negatively regulates PKB signaling pathways, impacting downstream targets like glycogen synthase kinase-3.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Protein kinase B (PKB), also known as Akt, is a crucial serine/threonine kinase activated by growth factors via phosphatidylinositol 3-kinase.
- Understanding PKB regulation is vital for comprehending cellular growth and survival pathways.
Purpose of the Study:
- To investigate the interaction between Protein Kinase B (PKB) and Protein Kinase C zeta (PKCzeta).
- To elucidate the role of PKCzeta in regulating PKB activity and downstream signaling.
Main Methods:
- Co-immunoprecipitation assays in CHO and COS-1 cell lines to detect PKB-PKCzeta complex formation.
- Site-directed mutagenesis to identify the interaction domain (AH domain of PKB).
- Analysis of kinase activity using kinase-inactive mutants and constitutively active mutants of PKB and PKCzeta.
- Assessment of downstream signaling pathways, including glycogen synthase kinase-3 inhibition.
Main Results:
- PKCzeta, but not PKCalpha or PKCdelta, co-immunoprecipitated with PKB.
- The association between PKB and PKCzeta is mediated by the AH domain of PKB.
- Platelet-derived growth factor (PDGF) stimulation decreased PKB-PKCzeta interaction, dependent on PKB activity.
- Co-expression of PKCzeta significantly abolished PDGF- and p110-CAAX-induced PKB activation.
- PKCzeta inhibited PKB's downstream signaling, including glycogen synthase kinase-3 inhibition.
Conclusions:
- PKB and PKCzeta interact in vivo.
- PKCzeta acts as a negative regulator of PKB activity.
- This interaction has significant implications for PKB-mediated cellular signaling pathways.