Dynamic Sequestration of the Recycling Compartment by Classical Protein Kinase C

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 m) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.The complement system is a major component of the innate immune system. It efficiently protects the host from pathogenic microorganisms, is involved in immune complex regulation, and serves an important link between innate and adaptive immune responses. Complement comprises a group of more than 30 proteins; most of them participate in the cascade-like activation process, whereas others serve as control proteins or act as cellular receptors (1, 2). Initiation of the complement activation cascade may occur through the classical, alternative, or lectin pathways. The three initiation pathways lead to activation of the terminal complement pathway and to assembly of the membrane attack complexes (MACs) 2 that are composed of the complement components C5b, C6, C7, C8, and C9 (also known as the C5b-9 complexes). Upon formation of C5b-7, C5b-8, and finally C5b-9 complexes, they adhere and then insert into the target cell membrane (3). The C5b-9 complexes may contain 1-18 C9 molecules attached to a C5b-8 complex. When the number of C9 molecules per C5b-8 exceeds 12, they self-polymerize and form a cylinder-shaped transmembrane structure (4). Upon its assembly, the MAC expresses neoantigens that can be detected by specific monoclonal antibodies (5). Although red blood cells require only one effective MAC to lyse, nucleated cells are killed by complement only after a combined action of several MACs (6). Although larger C5b-9 channels containing several C9 a...

Section: Discussionmentioning

confidence: 99%

Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

Moskovich

Fishelson

2007

“…Activity-A previous study implicated PKC in agonist-induced 5-HT 2A R internalization (20), and our work has established a critical role for cPKC in the sequestration of recycling molecules into a subset of endosomes in the perinuclear region (6,7). In addition, it was demonstrated previously that sustained activation of cPKC by PMA not only caused sequestration of recycling components but also led to a concomitant translocation of cPKC␣ and -␤II but not -␤I into the pericentrion (8).…”

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

confidence: 98%

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

Self Cite

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.

“…Evidence from our studies pointed that both the constitutive and the PKC-modulated OAT1 internalization occurred partly through a dynamin-and clathrin-dependent pathway. Indeed, treatment of OAT1-expressing cells with ConA, depletion of K ϩ from the cells, or transfection of dominant negative mutants of dynamin-2 into the cells, all of which blocked clathrin-dependent pathway (42)(43)(44)(45), significantly blocked OAT1 internalization (Figs. 8 -10).…”

Section: Discussionmentioning

confidence: 99%

“…To determine whether OAT1 internalizes via clathrin-dependent pathway, the constitutive and PKC-modulated OAT1 internalization were determined under the manipulations that specifically block clathrin-dependent internalization (42)(43)(44)(45): (i) treatment of the cells with concanavalin A (ConA) (Fig. 8a, top panel and Fig.…”

Section: Characterization Of Oat1 In Cos-7mentioning

confidence: 99%

Organic Anion Transporter OAT1 Undergoes Constitutive and Protein Kinase C-regulated Trafficking through a Dynamin- and Clathrin-dependent Pathway

Zhang

Hong

Duan

et al. 2008

121

Organic anion transporter 1 (OAT1) mediates the body disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We previously demonstrate that OAT1 activity was down-regulated by activation of protein kinase C (PKC), kinetically revealed as a decrease in the maximum transport velocity V max without significant change in the substrate affinity K m of the transporter. In the current study, we showed that OAT1 constitutively internalized from and recycled back to the plasma membrane, and PKC activation accelerated OAT1 internalization without affecting OAT1 recycling. We further showed that treatment of OAT1-expressing cells with concanavalin A, depletion of K ؉ from the cells, or transfection of dominant negative mutants of dynamin-2 or Eps15 into the cells, all of which block the clathrindependent endocytotic pathway, significantly blocked constitutive and PKC-regulated OAT1 internalization. We finally showed that OAT1 colocalized with transferrin, a marker for clathrin-dependent endocytosis, at the cell surface and in the EEA1-positive early endosomes. Together, our findings demonstrated for the first time that (i) OAT1 constitutively traffics between plasma membrane and recycling endosomes, (ii) PKC activation down-regulates OAT1 activity by altering already existent OAT1 trafficking, and (iii) OAT1 internalization occurs partly through a dynamin-and clathrin-dependent pathway.