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 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.
Hansra
G
G
Imperial Cancer Research Fund, Protein Phosphorylation Laboratory, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.
Garcia-Paramio
P
P
Prevostel
C
C
Whelan
R D
RD
Bornancin
F
F
Parker
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020298:U937 Cells
D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha