Abstract
Protein kinase C signaling is desensitized through a combination of dephosphorylation and proteolysis in intact cells. The process of dephosphorylation is analyzed here, as well as its relationship to degradation. It is established for protein kinase Calpha that dephosphorylation occurs in a membrane compartment following activation and temporally preceding significant degradation. The phosphatase responsible for the dephosphorylation appears to be a heterotrimeric type 2A phosphatase, which is shown to be in part constitutively membrane associated. Consistent with a role for this activity, okadaic acid is shown to inhibit the phorbol ester-induced dephosphorylation of protein kinase C that occurs in intact cells. Furthermore, phorbol ester-induced down-regulation of protein kinase Calpha is shown not to be dependent on the rate of dephosphorylation, indicating that these desensitizing pathways may operate in parallel.
Highlights
Members of the protein kinase C (PKC)1 family constitute a class of diacylglycerol-dependent protein kinases
It is clear that in other contexts, chronic activation by TPA leads to the generation of dephosphorylated forms that migrate faster on SDS-polyacrylamide gel electrophoresis (PAGE); these forms usually accumulate in association with the cytoskeleton
This study describes the identification of a membrane-associated protein phosphatase 2A heterotrimer that acts on PKC␣ in vitro and in intact cells and further analyses the relationship between the proteolytic degradation and dephosphorylation of PKC␣
Summary
Members of the protein kinase C (PKC)1 family constitute a class of diacylglycerol-dependent protein kinases (reviewed in Refs. 1– 4). This study describes the identification of a membrane-associated protein phosphatase 2A heterotrimer that acts on PKC␣ in vitro and in intact cells and further analyses the relationship between the proteolytic degradation and dephosphorylation of PKC␣. Preliminary data indicated that dephosphorylation of COS cell PKC␣ could be detected within 60 min of TPA treatment (see below).
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