Protein kinase C-dependent inhibition of the lysosomal degradation of endocytosed proteins in rat hepatocytes

Larocca, M. Cecilia; Ochoa, Elena J.; Garay, Esperanza; Marinelli, Raúl A.

doi:10.1016/s0898-6568(02)00003-7

Cited by 10 publications

(8 citation statements)

References 34 publications

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“…The relation between PKC activation and autophagy is not clear, but PMA through PKCα activation has been shown to increase the volume of autophagosomes in hepatocytes [44]. In the same line, we have shown recently that the phorbol ester and PKC activator PMA promotes autophagy in K562 cells [45].…”

Section: Discussionmentioning

confidence: 91%

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

et al. 2009

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CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

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Section: Discussionmentioning

confidence: 91%

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

et al. 2009

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“…Although the role of MARCKS phosphorylation has been investigated in other cell types, little is known about its effect in hepatocytes. PMA has been shown to phosphorylate and translocate MARCKS to lysosome in rat hepatocytes 36. Studies in most other cell types suggest a role of MARCKS in the exocytosis and exocytotic insertion of membrane proteins.…”

Section: Discussionmentioning

confidence: 96%

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells

2013

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Taurolithocholate (TLC) acutely inhibits biliary excretion of Mrp2 substrates by inducing Mrp2 retrieval from the canalicular membrane, while cAMP increases plasma membrane MRP2. The effect of TLC may be mediated via protein kinase Cε (PKCε). Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is a membrane bound F-actin cross linking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis and the underlying mechanism appears to be detachment of phosphorylated MARCKS from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCε mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with NTCP (HuH-NTCP cells) and in rat hepatocytes. TLC increased plasma membrane (PM) PKCε and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. Cyclic AMP did not affect PM-PKCε and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant negative (DN) PKCε reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and PMA increased cytosolic phospho-MARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCε suggesting PKCε mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation deficient MARCKS, TLC failed to increase MARCKS phosphorylation and to decrease plasma membrane MRP2. Conclusion Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC mediated activation of PKCε followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.

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“…Furthermore, different isoforms of PKC have been located in intracellular structures related to intracellular trafficking such as in caveolae, along the structures of the endocytic compartment, the Golgi complex and in lysosomes. The role of PKC in endocytosis, sorting, and/or trafficking is not completely understood but its activity seems important (Sanchez et al, 1998;Bao et al, 2000;Prevostel et al, 2000;Larocca et al, 2002;Ridge et al, 2002; Becker and Hanunn, 2003). In the present study, the activated form of PKC␦ has been located in endosomal fractions isolated from COS cells, and by using inhibitors and/or the small interfering RNA (siRNA)-mediated down-regulation we demonstrate that in the absence of calmodulin, PKC␦ is specifically involved in the regulation of EGFR exit from the early endocytic compartment.…”

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confidence: 99%

Protein KinaseCδ-Calmodulin Crosstalk Regulates Epidermal Growth Factor Receptor Exit from Early Endosomes

Lladó

Tebar

Calvo

et al. 2004

MBoC

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We have recently shown that calmodulin antagonist W13 interferes with the trafficking of the epidermal growth factor receptor (EGFR) and regulates the mitogen-activated protein kinase (MAPK) signaling pathway. In the present study, we demonstrate that in cells in which calmodulin is inhibited, protein kinase C (PKC) inhibitors rapidly restore EGFR and transferrin trafficking through the recycling compartment, although onward transport to the degradative pathway remains arrested. Analysis of PKC isoforms reveals that inhibition of PKC␦ with rottlerin or its down-modulation by using small interfering RNA is specifically responsible for the release of the W13 blockage of EGFR trafficking from early endosomes. The use of the inhibitor Gö 6976, specific for conventional PKCs (␣, ␤, and ␥), or expression of dominant-negative forms of PKC, , or ⑀ did not restore the effects of W13. Furthermore, in cells treated with W13 and rottlerin, we observed a recovery of brefeldin A tubulation, as well as transport of dextran-fluorescein isothiocyanate toward the late endocytic compartment. These results demonstrate a specific interplay between calmodulin and PKC␦ in the regulation of the morphology of and trafficking from the early endocytic compartment. INTRODUCTIONThe early endosome is a highly complex and dynamic intracellular compartment involved in the sorting of endocytosed receptors and ligands, for receptor recycling or targeting to lysosomes; in addition, it participates in endosome-endosome fusion and fission events (Gruenberg, 2001). The identification of microdomains in early endosomes, together with specific molecular activities (i.e., phosphorylation of signaling proteins or ubiquitylation of receptors), suggests that sorting and exit (budding) from this compartment are finely regulated and further indicates that our knowledge of its molecular machinery is incomplete. Thus, in addition of proteins that might be involved in the formation of specific domains (domain organizers) such as Rab5, Rab4, or annexin 2, other components are also likely to be important for the integrated function of endosomal sorting and trafficking.In a previous study, we demonstrated the importance of calmodulin in the regulation of early endocytic compartment morphology as well as in the trafficking and signaling of the epidermal growth factor receptor (EGFR) in this structure (Tebar et al., 2002). Now, we analyze the molecular mechanisms and the function of calmodulin in EGFR exit from early endosomes.Calmodulin specifically interacts with the EGFR in a calcium-dependent manner (Martin-Nieto and Villalobo, 1998;Li et al., 2004). Mutations in the juxtamembrane domain (calmodulin-binding site) of the EGFR, or deletion of the basic segment (645-660 amino acids), inhibit this interaction; interestingly, this region contains the threonine 654, the target of protein kinase C (PKC). Calmodulin binding inhibits EGFR tyrosine kinase activity in vitro (San José et al., 1992) and PKC-induced phosphorylation at Thr654 (Lund et al., 1990;Bao et al., 20...

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Protein kinase C-dependent inhibition of the lysosomal degradation of endocytosed proteins in rat hepatocytes

Cited by 10 publications

References 34 publications

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells

Protein KinaseCδ-Calmodulin Crosstalk Regulates Epidermal Growth Factor Receptor Exit from Early Endosomes

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