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Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes
G Hansra1, P Garcia-Paramio, C Prevostel
1Imperial Cancer Research Fund, Protein Phosphorylation Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
Abstract:
The generation of antisera specific for the priming phosphorylation sites on protein kinase Calpha (PKCalpha) has permitted analysis of the dephosphorylation of these sites in relation to the down-regulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKCalpha is able to stimulate the dephosphorylation of wild-type PKCalpha in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKCalpha on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKCalpha is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKCbeta1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.
Insights
Protein kinase Calpha (PKCalpha) undergoes agonist-induced dephosphorylation at priming sites, indicating inactivation. This PKC-dependent process requires catalytic activity and involves membrane trafficking, suggesting a general desensitization mechanism for conventional PKCs.
Area of Science:
- Cellular signaling
- Enzymology
- Molecular biology
Background:
- Protein kinase Calpha (PKCalpha) plays a crucial role in cellular signaling pathways.
- Understanding the regulation and desensitization of PKCalpha is vital for comprehending its physiological functions.
- Priming phosphorylation sites are key regulatory elements in PKCalpha activation.
Purpose of the Study:
- To investigate the dephosphorylation of priming phosphorylation sites on PKCalpha.
- To elucidate the relationship between dephosphorylation, PKC catalytic activity, and protein down-regulation.
- To determine if this dephosphorylation is a general desensitization mechanism for conventional protein kinase C isotypes.
Main Methods:
- Generation of specific antisera for PKCalpha priming phosphorylation sites.
- Analysis of dephosphorylation in response to agonists and PKC inhibitors.
- Utilizing constitutively active PKCalpha fragments in transfected cells.
- Investigating temperature sensitivity and cellular localization (cytoplasmic vesicles) of the dephosphorylation process.
Main Results:
- Agonist-induced dephosphorylation of PKCalpha priming sites was observed, correlating with protein inactivation.
- PKC catalytic activity is required for this dephosphorylation, as shown by inhibitor studies and constitutively active fragments.
- The dephosphorylation process is temperature-sensitive and involves transient accumulation of PKCalpha on cytoplasmic vesicular structures, suggesting membrane trafficking.
- Dephosphorylation of priming sites was also observed in other conventional PKC isotypes (e.g., PKCbeta1), indicating a conserved desensitization mechanism.
- Dephosphorylation of the PKCbeta1 activation loop site was shown to be cell density-dependent in U937 cells, highlighting physiological relevance.
Conclusions:
- Agonist stimulation leads to PKCalpha inactivation via dephosphorylation of priming sites, dependent on PKC catalytic activity.
- This dephosphorylation is linked to a temperature-sensitive membrane trafficking event.
- The findings reveal a general desensitization mechanism for conventional PKC isotypes, crucial for regulating kinase activity.
- Cell density-dependent dephosphorylation of PKCbeta1 underscores the physiological significance of this desensitization process.