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

2004 ◽  
Vol 15 (11) ◽  
pp. 4877-4891 ◽  
Author(s):  
Anna Lladó ◽  
Francesc Tebar ◽  
Maria Calvo ◽  
Jemina Moretó ◽  
Alexander Sorkin ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Calmodulin Antagonist ◽  
Endocytic Compartment ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
In Cells

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 PKCδ 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 (α, β, and γ), or expression of dominant-negative forms of PKCλ, ζ, or ϵ 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 PKCδ in the regulation of the morphology of and trafficking from the early endocytic compartment.

scholarly journals Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

2008 ◽  
Vol 19 (1) ◽  
pp. 17-29 ◽  
Author(s):  
Anna Lladó ◽  
Paul Timpson ◽  
Sandra Vilà de Muga ◽  
Jemina Moretó ◽  
Albert Pol ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Intracellular Trafficking ◽  
Actin Polymerization ◽  
Growth Factor Receptor ◽  
Early Endosomes ◽  
Epidermal Growth ◽  
Protein Kinase Cδ

The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR.

scholarly journals A Role for Sorting Nexin 2 in Epidermal Growth Factor Receptor Down-regulation: Evidence for Distinct Functions of Sorting Nexin 1 and 2 in Protein Trafficking

2004 ◽  
Vol 15 (5) ◽  
pp. 2143-2155 ◽  
Author(s):  
Anuradha Gullapalli ◽  
Tiana A. Garrett ◽  
May M. Paing ◽  
Courtney T. Griffin ◽  
Yonghua Yang ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Protein Trafficking ◽  
Growth Factor Receptor ◽  
Down Regulation ◽  
Sorting Nexin ◽  
Px Domain ◽  
Early Endosomes ◽  
Epidermal Growth

Sorting nexin 1 (SNX1) and SNX2, homologues of the yeast vacuolar protein-sorting (Vps)5p, contain a phospholipid-binding motif termed the phox homology (PX) domain and a carboxyl terminal coiled-coil region. A role for SNX1 in trafficking of cell surface receptors from endosomes to lysosomes has been proposed; however, the function of SNX2 remains unknown. Toward understanding the function of SNX2, we first examined the distribution of endogenous protein in HeLa cells. We show that SNX2 resides primarily in early endosomes, whereas SNX1 is found partially in early endosomes and in tubulovesicular-like structures distributed throughout the cytoplasm. We also demonstrate that SNX1 interacts with the mammalian retromer complex through its amino terminal domain, whereas SNX2 does not. Moreover, activated endogenous epidermal growth factor receptor (EGFR) colocalizes markedly with SNX2-positive endosomes, but minimally with SNX1-containing vesicles. To assess SNX2 function, we examined the effect of a PX domain-mutated SNX2 that is defective in vesicle localization on EGFR trafficking. Mutant SNX2 markedly inhibited agonist-induced EGFR degradation, whereas internalization remained intact. In contrast, SNX1 PX domain mutants failed to effect EGFR degradation, whereas a SNX1 deletion mutant significantly inhibited receptor down-regulation. Interestingly, knockdown of SNX1 and SNX2 expression by RNA interference failed to alter agonist-induced EGFR down-regulation. Together, these findings suggest that both SNX1 and SNX2 are involved in regulating lysosomal sorting of internalized EGFR, but neither protein is essential for this process. These studies are the first to demonstrate a function for SNX2 in protein trafficking.

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