Isoenzyme-specific Translocation of Protein Kinase C (PKC)βII and not PKCβI to a Juxtanuclear Subset of Recycling Endosomes

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Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. It is also well established that ligand stimulation of many plasma membrane receptors leads to their internalization. However, stimulus-induced regulation of endosomal trafficking has not received much attention. In previous studies, we showed that sustained stimulation of protein kinase C (PKC) with phorbol esters led to sequestration of recycling endosomes in a juxtanuclear region. In this study, we investigated whether G-protein-coupled receptors that activate PKC exerted effects on endosomal trafficking. Stimulation of cells with serotonin (5-hydroxytryptamine (5-HT)) led to sequestration of the 5-HT receptor (5-HT 2A R) into a Rab11-positive juxtanuclear compartment. This sequestration coincided with translocation of PKC as shown by confocal microscopy. Mechanistically the observed sequestration of 5-HT 2A R was shown to require continuous PKC activity because it was inhibited by pretreatment with classical PKC inhibitor Gö6976 and could be reversed by posttreatment with this inhibitor. In addition, classical PKC autophosphorylation was necessary for receptor sequestration. Moreover inhibition of phospholipase D (PLD) activity and inhibition of PLD1 and PLD2 using dominant negative constructs also prevented this process. Functionally this sequestration did not affect receptor desensitization or resensitization as measured by intracellular calcium increase. However, the PKC-and PLD-dependent sequestration of receptors resulted in co-sequestration of other plasma membrane proteins and receptors as shown for epidermal growth factor receptor and protease activated receptor-1. This led to heterologous desensitization of those receptors and diverted their cellular fate by protecting them from agonist-induced degradation. Taken together, these results demonstrate a novel role for sustained receptor stimulation in regulation of intracellular trafficking, and this process requires sustained stimulation of PKC and PLD.

Section: Sequestration Of 5-ht 2a Receptors Coincides With Sequestratmentioning

confidence: 83%

“…shown previously that PLD activation was required for translocation of PKC␣/␤II and formation of the pericentrion (8). To further define the mechanism of receptor sequestration, it became important to determine whether the PLD pathway was involved in agonist-induced 5-HT 2A R sequestration.…”

Section: Pld Activity Is Required For Receptor Sequestration-it Wasmentioning

confidence: 99%

Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner

Idkowiak‐Baldys¹,

Baldys

Raymond

et al. 2009

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“…Moreover, sustained activation of PKC also resulted in sequestration and retention of recycling transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. Mechanistically, this sequestration was shown to be phospholipase D-dependent (8,9) and was also selectively inhibited by a mechanism involving ceramide formed from the salvage pathway (10).…”

Section: Members Of the Protein Kinase C (Pkc)mentioning

confidence: 99%

Dynamic Sequestration of the Recycling Compartment by Classical Protein Kinase C

Idkowiak‐Baldys

Becker

Kitatani

et al. 2006

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It has been previously shown that upon sustained stimulation (30 -60 min) with phorbol esters, protein kinase C (PKC) ␣ and ␤II 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°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.

“…This novel translocation is seen with prolonged stimulation of these two isoenzymes of PKC by PMA (for at least 30 min) and follows the initial translocation of PKC to the plasma membrane (seen as early as 2-5 min following stimulation with PMA). Most interestingly, this novel translocation operates selectively on PKC ␣ and ␤II in that it is not observed with PKC ␤I or any of the nonclassical PKCs (1,6). Mechanistically, this translocation involves activation of PLD (6) and the interaction of PKC with PLD (5).…”

mentioning

confidence: 99%

Selective Inhibition of Juxtanuclear Translocation of Protein Kinase C βII by a Negative Feedback Mechanism Involving Ceramide Formed from the Salvage Pathway

Becker

Kitatani

Idkowiak‐Baldys

et al. 2005

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In a previous study, we showed that protein kinase C ␤II (PKC ␤II) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC ␤II with 4␤-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC ␤II. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC ␤II in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC ␤II in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC ␤II in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC ␤II.The isoenzymes of protein kinase C (PKC), 1 designated as the classical PKCs, comprise a subfamily of closely related isoenzymes that share similar structural features and mechanisms of action and regulation. This group consists of PKC ␣, PKC ␤I, PKC ␤II, and PKC ␥ that share highly homologous regulatory domains and are activated by diacylglycerol (DAG) and calcium. Thus, they serve as signal transducers for the action of extracellular agonists that activate phospholipases C, which results in the generation of DAG and calcium (2, 3).Activation of PKC requires the translocation of PKC to the plasma membrane in response to acutely generated DAG (3, 4). This translocation also places PKC in proximity to specific membrane substrates. Thus, the dynamic interaction of PKC with membranes has emerged as a key mechanism in regulating its function.Previous studies from our laboratory (1), supported by the recent results from Hu and Exton (5), have demonstrated a novel translocation of PKC ␣ and ␤II to a juxtanuclear region. This novel translocation is seen with prolonged stimulation of these two isoenzymes of PKC by PMA (for at least 30 min) and follows the initial translocation of ...