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Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32
1Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Abstract:
We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.
Insights
The multifunctional chaperon protein p32 binds to protein kinase C mu (PKC mu) at mitochondrial membranes. This interaction regulates PKC mu
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Protein-Protein Interactions
Background:
- Protein kinase C (PKC) enzymes play crucial roles in cellular signaling.
- The multifunctional chaperon protein p32 is involved in various cellular processes.
- The specific interactions and regulatory roles of p32 with different PKC isoforms are not fully understood.
Purpose of the Study:
- To identify and characterize the interaction between the chaperon protein p32 and protein kinase C mu (PKC mu).
- To investigate the functional consequences of the p32-PKC mu interaction on PKC mu kinase activity.
- To determine the subcellular localization and specificity of the p32-PKC mu association.
Main Methods:
- Yeast two-hybrid analysis to detect in vivo protein-protein interactions.
- Glutathione S-transferase (GST) pull-down assays to confirm binding.
- Reciprocal co-immunoprecipitation experiments in SKW 6.4 and 293T cell lines.
- Subcellular fractionation and colocalization studies with cytochrome c.
- In vitro kinase assays using aldolase as a substrate.
Main Results:
- p32 was identified as a binding protein for multiple PKC isoforms, including PKC mu.
- In vivo association of PKC mu and p32 was confirmed through multiple biochemical methods.
- PKC mu and p32 constitutively associate at mitochondrial membranes in a compartment-specific manner.
- p32 binds to the kinase domain of PKC mu but does not act as a substrate.
- p32 binding inhibits PKC mu's ability to phosphorylate substrates like aldolase in vitro, suggesting a regulatory role.
Conclusions:
- p32 is a novel, compartment-specific regulator of PKC mu kinase activity.
- The interaction between p32 and PKC mu occurs at mitochondrial membranes and modulates substrate phosphorylation.
- These findings reveal a new mechanism for controlling PKC signaling pathways.