Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes

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...

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Bibliographic Details
Published in:Biochemical journal 1999-09, Vol.342 ( Pt 2) (2), p.337-344
Main Authors: Hansra, G, Garcia-Paramio, P, Prevostel, C, Whelan, R D, Bornancin, F, Parker, P J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites - genetics
COS Cells
Green Fluorescent Proteins
Humans
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - metabolism
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Molecular Sequence Data
Phosphorylation
Protein Kinase C - chemistry
Protein Kinase C - genetics
Protein Kinase C - metabolism
Protein Kinase C-alpha
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Sequence Homology, Amino Acid
Transfection
U937 Cells
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Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/0264-6021:3420337