Protein kinase Cdelta 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 Cdelta (PKCdelta). On inhibition of CaM, PKCdelta promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCdelta impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCdelta-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCdelta. 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 PKCdelta, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCdelta organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR.

Figure 1.

CaM regulates F-actin dynamics in early endosomes. (A) NRK cells were incubated for 60 min with the specific CaM inhibitor W13 (5 μg/ml) at 37°C. Labeling with phalloidin-TRITC and anti-EEA1 (Alexa Fluor 488) is shown. (B) High magnification of actin patches surrounding endosomes of cells treated with W13 as described in A. (C) NRK cells were microinjected with YFP-actin, DNA expression vector, and incubated 60 min with W13 (5 μg/ml) and 30 min with transferrin-TRITC at 37°C. Bars, 10 μm.

Figure 2.

Actin may control early endosome exit in cells treated with the CaM antagonist. (A) NRK cells were incubated for 45 min with W13 (5 μg/ml) and 30 min with the actin-depolymerizing agent cytochalasin D (1 μM). Anti-EEA1 (Alexa Fluor 488) and phalloidin-TRITC stainings are shown. Bar, 10 μm. (B) Vero cells transiently expressing constitutively active YFP-cofilin (S3A) were treated with W13 for 60 min at 37°C. After fixation, the cells were immunolabeled with anti-EEA1 (Alexa Fluor 594) and phalloidin-TRITC. Bar, 10 μm.

Figure 3.

A CaM and PKCδ interplay modulates actin on endosomes. NRK cells were preincubated with W13 (5 μg/ml) for 30 min, treated with the PKCδ-specific inhibitor rottlerin (5 μM) for 30 min, and then 15 min with transferrin-TRITC (50 μg/ml) at 37°C. EEA1 (Alexa Fluor 488), transferrin-TRITC, and phalloidin (Alexa Fluor 350) stainings are shown. Bar, 10 μm.

Figure 4.

Comparison of RhoB and CaM roles in endosomes. (A) Twenty-four hours after transfection with myc-RhoB wt, COS1 cells were incubated with EGF (100 ng/ml; 15 min) at 37°C, fixed, and triple-stained with anti-myc (Alexa Fluor 488), phalloidin-TRITC and anti-EGFR (Cy5). (B) COS1 cells were transfected with pSUPER RhoB, and, after 72 h of knockdown, cells were incubated with W13 (5 μg/ml; 60 min) at 37°C. EEA1 was detected with specific antibody followed by Alexa Fluor 594. The extent of RhoB depletion is shown by Western blotting. (C) NRK cells were preincubated with W13 (5 μg/ml; 30 min), treated with the ROCK inhibitor Y27632 (20 μM) for the next 45 min and with transferrin-TRITC (50 μg/ml) for the last 15 min at 37°C. After fixation, cells were labeled with phalloidin-Alexa Fluor 350 and anti-EEA1 (Alexa Fluor 488). Bar, 10 μm. (D) COS1 cells were transfected and treated as described in A, adding 5 μM rottlerin for 30 min. Bar, 10 μm. On the right, quantification of the number of RhoB-overexpressing cells presenting 5 or more enlarged endosomes (diameter >350 nm) from two independent experiments (performed as described above; n = 900 cells) is represented. Each data point represents value averaged (±SD). ***p < 0.001 for Student's t test, indicating statistical significance of differences between EGF- and EGF- + rottlerin-treated cells.

Figure 5.

Arp2/3 participates in the CaM and PKCδ regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with GFP (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.

Figure 6.

N-WASP or WAVE are not involved in W13 effect. (A) COS1 cells transiently expressing GFP-N-WASP-ΔWA were incubated with W13 (5 μg/ml) for 90 min and EGF (100 ng/ml) for the last 15 min at 37°C, fixed, and immunolabeled with anti-EGFR (Alexa Fluor 594). Bar, 10 μm. (B) NRK cells were preincubated with W13 (7.5 μg/ml) for 60 min and then treated with transferrin-TRITC (50 μg/ml) for 30 min at 37°C. Next, cells were fixed and stained with anti-WAVE (Alexa Fluor 488). Bar, 10 μm.

Figure 7.

Cortactin regulates recycling in the early endosomes. (A) NRK cells were preincubated for 30 min with W13 (5 μg/ml), treated with rottlerin (5 μM) for 30 min, and then with transferrin (50 μg/ml) for 15 min at 37°C. Cortactin (Alexa Fluor 488) and transferrin-TRITC staining are shown. Bar, 10 μm. (B) HeLa cells were transfected for 72 h with cortactin siRNA duplex 1 and 2 or GFP siRNA. Then, cells internalized ¹²⁵I-EGF (5 ng/ml; 7 min), and they were treated with W13 (7.5 μg/ml) for 30 min at 37°C to measure recycling and degradation. Each data point in the histogram represents the mean of a minimum of six replicates from two independent experiments for each siRNA (±SD). *p < 0.05, **p < 0.01, ***p < 0.001 for Student's t test, indicating statistical significances of differences between GFP and cortactin siRNA-transfected cells. The degree of down-regulation of cortactin is demonstrated by Western blotting.

Figure 8.

CaM and PKCδ interact with cortactin. (A) Cellular lysates from HeLa cells were incubated with CaM-Sepharose in the presence of Ca²⁺ or EGTA as described in Materials and Methods. All bound fraction and 25 μl of the unbound fraction or lysate was loaded and the presence of cortactin and RhoB was analyzed by Western blotting. (B) HeLa cells were cotransfected with cortactin and GFP or GFP-PKCδ. After 24-h transfection, cells were incubated for 30 min with or without W13 (10 μg/ml) at 37°C. HeLa cell extracts were incubated with anti-GFP polyclonal antibodies, and the immunocomplex was pulled down by using protein A-Sepharose. The presence of cortactin and GFP or GFP-PKCδ in the immunoprecipitates was analyzed by Western blotting by using anti-cortactin and anti-GFP antibodies, respectively. (C) HeLa cells were treated for 30 min ± W13 (10 μg/ml) at 37°C. Cell extracts were incubated with anti-cortactin monoclonal antibody and pulled down using protein G-Sepharose. The presence of PKCδ and cortactin in the immunoprecipitates was analyzed by Western blotting.