Dynamic sequestration of the recycling compartment by classical protein kinase C.
It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 degrees C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.
Idkowiak-Baldys
Jolanta
J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Becker
Kevin P
KP
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Kitatani
Kazuyuki
K
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425.
Hannun
Yusuf A
YA
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425. Electronic address: hannun@musc.edu.
D018957:CD59 Antigens / Q000378:metabolism; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D018957:CD59 Antigens; D002966:Clathrin; D014168:Transferrin; C051590:CD59 protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate