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

Abstract

The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase C␦ (PKC␦). On inhibition of CaM, PKC␦ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKC␦ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKC␦-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKC␦. 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 PKC␦, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKC␦ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. INTRODUCTIONCalmodulin (CaM) is a ubiquitous small calcium sensor that regulates a variety of cellular processes in a spatial and temporal manner within the cell (Toutenhoofd and Strehler, 2000). Microenvironment variations in the concentration of CaM may be critical to the control of cellular processes that involve specific and high-affinity CaM-binding proteins (Berridge et al., 2000;Carafoli, 2002;Kahl and Means, 2003).We have previously shown that CaM is crucial to the regulation of the dynamics of endosomes. In particular, in the presence of W13, a highly specific CaM antagonist, trafficking is blocked in early endosomes, thereby interfering with transport toward degradation and recycling pathways. This blockage was associated with a dramatic alteration of the morphology and size of early endosomes (Tebar et al., 2002;Lladó et al., 2004). Furthermore we showed that a cross-talk between CaM and protein kinase C␦ (PKC␦) is involved in the regulation of the budding from the early endocytic compartment (Lladó et al., 2004).Several lines of evidence implicate the actin cytoskeleton in the CaM-and PKC␦-mediated regulation of vesicular trafficking. Because actin contributes to the maintenance of organelle stability (Apodaca, 2001), swelling and morphological changes observed in response to CaM/PKC␦ activity (Lladó et al., 2004) may involve altered actin dynamics. Moreover, several CaM-binding proteins and PKC␦ substrates are also actin-binding proteins, including ␣-actinin, adducin, and myristoylated alanine-rich protein kinase C substrate (MARCKS) (Chakravarthy et al., 1999). Finally, RhoGTPases and CaM-binding proteins responsible for fusion-fission events during vesicular trafficking also depend on actin filaments for pinch-off and vesicular movement (Mayorga et al., 1994;Mu et al., 1995;Colombo et al., 1997;Ridley, 2001;Burgoyne and Clague, 2003;Lawe et al., 2003;Donaldson, 2005).The actin cytosk...

Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

“…Consequently, internalized pThr654-EGF receptor is not targeted to lysosomes but diverted to recycling endosomes for transport back to the cell surface (96,97). EGF receptor recycling was previously considered a default pathway, but we showed, that this involves PKCa-dependent regulation of actin dynamics in the early endosomal compartment (74,75). Thus AnxA6-induced Thr654 EGF receptor phosphorylation may link actin and PKCd to EGF receptor recycling.…”

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A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells

“…As endocytic recycling of ErbB2 is thought to critically contribute to its oncogenic signaling (18,80), and enhanced lysosomal degradation of ErbB2 is seen with ErbB2-targeted therapeutics (10,78), a better understanding of the biochemical pathways that regulate ErbB2 recycling is of substantial interest. Analyses of the related family member EGFR suggest an important role of cellular kinases (62). However, the role of protein kinases in ErbB2 recycling is unclear.…”

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

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