Dephosphorylation of PKC, either in vitro or in LTP samples, requires the presence of cofactors normally serving to activate PKC (i.e., Ca+2/PS/DAG). These observations lead to the hypothesis that phosphatase accessibility of phosphorylation sites on PKC is conformation-dependent. Sweatt et al. (46) tested this hypothesis by examining the phosphatase sensitivity of PKC, autophosphorylated with 32P-ATP in vitro, in the presence and absence of its activators, PS/DAG, and Ca+2. The presence of activators dramatically increased the rate of dephosphorylation of autophosphorylated PKC (see Panels A and B). In the presence of activators PKC was 83% dephosphorylated after incubation with protein phosphatase for 2 minutes, whereas in controls PKC was only 21% dephosphorylated after the 2-minute phosphatase treatment. Likewise, after a 30-minute incubation with phos-phatase, PKC incubated in the presence of activators was completely dephosphory-lated while control incubations exhibited notable phosphatase resistance. Western blot analysis with a C-terminal domain autophosphorylation-sensitive antiserum revealed that the rate of 32P release parallels the rate at which immunoreactivity decreased. Thus, dephosphorylation of PKC is markedly stimulated in the membrane-bound "activated" conformation of PKC.
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