An essential role for autophosphorylation in the dissociation of activated protein kinase C from the plasma membrane.
The cellular localization of protein kinase C (PKC) is intimately associated with the regulation of its biological activity. Previously we have demonstrated that the redistribution of PKC to the plasma membrane in response to physiological stimuli is followed by a rapid returning of PKC back to the cytoplasm (Feng, X., Zhang, J., Barak, L. S., Meyer, T., Caron, M. G., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 10755-10762). Although the process of PKC membrane targeting has been extensively studied, the molecular mechanism underlying the dissociation of membrane-bound PKC remains unclear. In the present study, by examining the dynamic distribution of wild-type PKC betaII and its kinase-deficient mutant (K371R), we demonstrate that kinase activity is required for PKC membrane dissociation. Moreover, the inability of PKC betaII(K371R) to dissociate from the plasma membrane in cells overexpressing wild-type PKC betaII suggests that autophosphorylation activity of the kinase might be essential for its membrane dissociation. This was further supported by mutational analysis of two in vivo autophosphorylation sites on PKC betaII. The replacement of Ser660 or Thr641 by alanine (S660A or T641A) was found to synergistically reduce the reversal of PKC betaII membrane translocation, whereas the replacement of the same amino acids by glutamic acid (S660E or T641E), an amino acid commonly used to mimic phosphate, results in mutants behaving similar to wild-type PKC betaII. These findings point to an essential role for autophosphorylation in the dissociation of activated PKC from the plasma membrane and suggest that, like PKC membrane translocation, the returning of PKC to the cytoplasm after its activation is also delicately regulated.
Feng
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X
Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Hannun
Y A
YA
D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta
D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta