Limits...
Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.

Hanafusa H, Ishikawa K, Kedashiro S, Saigo T, Iemura S, Natsume T, Komada M, Shibuya H, Nara A, Matsumoto K - Nat Commun (2011)

Bottom Line: Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments.Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes.Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate school of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

ABSTRACT
Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments. In this paper, we demonstrate that leucine-rich repeat kinase LRRK1, which contains a MAPKKK-like kinase domain, forms a complex with activated EGFR through an interaction with Grb2. Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes. LRRK1 regulates EGFR transport from early to late endosomes and regulates the motility of EGF-containing early endosomes in a manner dependent on its kinase activity. Furthermore, LRRK1 serves as a scaffold facilitating the interaction of EGFR with the endosomal sorting complex required for transport-0 complex, thus enabling efficient sorting of EGFR to the inner vesicles of multivesicular bodies. Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.

No MeSH data available.


Related in: MedlinePlus

Effects of LRRK1 depletion on localization of EGFR in MVBs and EGFR degradation.(a, b) Effect of LRRK1 depletion on EGFR sorting into the lumen of Rab5(Q79L)-induced enlarged endosomes. HeLa S3 cells treated with control (a) or LRRK1 siRNA (Stealth#1) (b) were transfected with DsRed-Rab5(Q79L) and stimulated with 100 ng per ml of Alexa 647-EGF for 30 min. The cells were fixed and imaged by confocal microscopy. Images are three-dimensional reconstructions from a series of confocal Z-stack images (0.3 μm-thick sections). Scale bar, 10 μm. (c, d) Confocal images from control (c; the boxed region in a) or LRRK1-depleted cells (d; the boxed region in b) show individual enlarged endosomes. Areas of co-localization are shown by line intensity profiles. (e) Quantification of Alexa 647-EGF localization into the lumen of Rab5(Q79L)-induced enlarged endosomes. Cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with DsRed-Rab5(Q79L) and siRNA-resistant GFP-LRRK1 (wild type and the K1243 M mutant), as indicated. Cells were stimulated with 100 ng per ml of Alexa 647-EGF for 30 min and then fixed. Data are presented as percentages of Alexa 647-EGF localization into the lumen of enlarged endosomes out of the total number of Rab5(Q79L)-induced enlarged endosomes (diameter; >1 μm). Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 100 endosomes) scored per experiment. (f) HeLa S3 cells were treated with control or LRRK1 siRNA (Stealth#1). After 16 h of serum starvation, cells were incubated with anti-EGFR antibodies (LA-22) for 2 h at 37°C, followed by goat anti-mouse IgM antibodies conjugated to 10 nm gold for 1 h at 37°C. After washing to remove antibodies from the medium, the cells were stimulated with EGF for 10 min and then fixed. The localization of EGFR was examined by silver-enhanced immunogold electron microscopy. Scale bar, 100 nm. (g) HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with EGFR. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Cell lysates were immunoblotted with indicated antibodies. Tubulin serves as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105304&req=5

f8: Effects of LRRK1 depletion on localization of EGFR in MVBs and EGFR degradation.(a, b) Effect of LRRK1 depletion on EGFR sorting into the lumen of Rab5(Q79L)-induced enlarged endosomes. HeLa S3 cells treated with control (a) or LRRK1 siRNA (Stealth#1) (b) were transfected with DsRed-Rab5(Q79L) and stimulated with 100 ng per ml of Alexa 647-EGF for 30 min. The cells were fixed and imaged by confocal microscopy. Images are three-dimensional reconstructions from a series of confocal Z-stack images (0.3 μm-thick sections). Scale bar, 10 μm. (c, d) Confocal images from control (c; the boxed region in a) or LRRK1-depleted cells (d; the boxed region in b) show individual enlarged endosomes. Areas of co-localization are shown by line intensity profiles. (e) Quantification of Alexa 647-EGF localization into the lumen of Rab5(Q79L)-induced enlarged endosomes. Cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with DsRed-Rab5(Q79L) and siRNA-resistant GFP-LRRK1 (wild type and the K1243 M mutant), as indicated. Cells were stimulated with 100 ng per ml of Alexa 647-EGF for 30 min and then fixed. Data are presented as percentages of Alexa 647-EGF localization into the lumen of enlarged endosomes out of the total number of Rab5(Q79L)-induced enlarged endosomes (diameter; >1 μm). Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 100 endosomes) scored per experiment. (f) HeLa S3 cells were treated with control or LRRK1 siRNA (Stealth#1). After 16 h of serum starvation, cells were incubated with anti-EGFR antibodies (LA-22) for 2 h at 37°C, followed by goat anti-mouse IgM antibodies conjugated to 10 nm gold for 1 h at 37°C. After washing to remove antibodies from the medium, the cells were stimulated with EGF for 10 min and then fixed. The localization of EGFR was examined by silver-enhanced immunogold electron microscopy. Scale bar, 100 nm. (g) HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with EGFR. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Cell lysates were immunoblotted with indicated antibodies. Tubulin serves as a loading control.

Mentions: EGFR localized in clathrin-coated microdomains on endosomes is sorted into intraluminal vesicles (ILVs) within MVBs and delivered to lysosomes for degradation. We therefore examined whether LRRK1 knockdown affects the sorting of EGFR into ILVs of enlarged endosomes using three-dimensional reconstructions. In control siRNA-transfected cells, Alexa 647-EGF was translocated from the limiting membrane to ILVs of Rab5(Q79L)-induced enlarged endosomes at 30 min after EGF stimulation (Fig. 8a). In contrast, depletion of LRRK1 reduced the intraluminal transport of Alexa 647-EGF into enlarged endosomes (Fig. 8b). Line intensity profiles of individual endosomes clearly showed that most of the EGF was localized within the inner membranes in control cells (Fig. 8c), but were retained in the limiting membrane in LRRK1-depleted cells (Fig. 8d). Quantification of EGF labelling showed that about 66.2% of the labelling was localized to the inner membranes in cells treated with control siRNA, whereas only 16.7% showed this localization in cells treated with LRRK1 siRNA (Fig. 8e). These results demonstrate that LRRK1 is needed for sorting of EGF/EGFR into ILVs of MVBs. When siRNA-resistant, wild-type GFP-LRRK1 was expressed in LRRK1-depleted cells, the defect in the intraluminal transport of Alexa 647-EGF was partially rescued (Fig. 8e). However, expression of the siRNA-resistant, kinase-negative GFP-LRRK1(K1243M) was able to rescue the defect in EGF sorting in LRRK1-depleted cells (Fig. 8e), suggesting that LRRK1 kinase activity is not essential for this step. Thus, LRRK1 functions as a scaffold in this process, independent of its kinase activity.


Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.

Hanafusa H, Ishikawa K, Kedashiro S, Saigo T, Iemura S, Natsume T, Komada M, Shibuya H, Nara A, Matsumoto K - Nat Commun (2011)

Effects of LRRK1 depletion on localization of EGFR in MVBs and EGFR degradation.(a, b) Effect of LRRK1 depletion on EGFR sorting into the lumen of Rab5(Q79L)-induced enlarged endosomes. HeLa S3 cells treated with control (a) or LRRK1 siRNA (Stealth#1) (b) were transfected with DsRed-Rab5(Q79L) and stimulated with 100 ng per ml of Alexa 647-EGF for 30 min. The cells were fixed and imaged by confocal microscopy. Images are three-dimensional reconstructions from a series of confocal Z-stack images (0.3 μm-thick sections). Scale bar, 10 μm. (c, d) Confocal images from control (c; the boxed region in a) or LRRK1-depleted cells (d; the boxed region in b) show individual enlarged endosomes. Areas of co-localization are shown by line intensity profiles. (e) Quantification of Alexa 647-EGF localization into the lumen of Rab5(Q79L)-induced enlarged endosomes. Cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with DsRed-Rab5(Q79L) and siRNA-resistant GFP-LRRK1 (wild type and the K1243 M mutant), as indicated. Cells were stimulated with 100 ng per ml of Alexa 647-EGF for 30 min and then fixed. Data are presented as percentages of Alexa 647-EGF localization into the lumen of enlarged endosomes out of the total number of Rab5(Q79L)-induced enlarged endosomes (diameter; >1 μm). Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 100 endosomes) scored per experiment. (f) HeLa S3 cells were treated with control or LRRK1 siRNA (Stealth#1). After 16 h of serum starvation, cells were incubated with anti-EGFR antibodies (LA-22) for 2 h at 37°C, followed by goat anti-mouse IgM antibodies conjugated to 10 nm gold for 1 h at 37°C. After washing to remove antibodies from the medium, the cells were stimulated with EGF for 10 min and then fixed. The localization of EGFR was examined by silver-enhanced immunogold electron microscopy. Scale bar, 100 nm. (g) HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with EGFR. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Cell lysates were immunoblotted with indicated antibodies. Tubulin serves as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105304&req=5

f8: Effects of LRRK1 depletion on localization of EGFR in MVBs and EGFR degradation.(a, b) Effect of LRRK1 depletion on EGFR sorting into the lumen of Rab5(Q79L)-induced enlarged endosomes. HeLa S3 cells treated with control (a) or LRRK1 siRNA (Stealth#1) (b) were transfected with DsRed-Rab5(Q79L) and stimulated with 100 ng per ml of Alexa 647-EGF for 30 min. The cells were fixed and imaged by confocal microscopy. Images are three-dimensional reconstructions from a series of confocal Z-stack images (0.3 μm-thick sections). Scale bar, 10 μm. (c, d) Confocal images from control (c; the boxed region in a) or LRRK1-depleted cells (d; the boxed region in b) show individual enlarged endosomes. Areas of co-localization are shown by line intensity profiles. (e) Quantification of Alexa 647-EGF localization into the lumen of Rab5(Q79L)-induced enlarged endosomes. Cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with DsRed-Rab5(Q79L) and siRNA-resistant GFP-LRRK1 (wild type and the K1243 M mutant), as indicated. Cells were stimulated with 100 ng per ml of Alexa 647-EGF for 30 min and then fixed. Data are presented as percentages of Alexa 647-EGF localization into the lumen of enlarged endosomes out of the total number of Rab5(Q79L)-induced enlarged endosomes (diameter; >1 μm). Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 100 endosomes) scored per experiment. (f) HeLa S3 cells were treated with control or LRRK1 siRNA (Stealth#1). After 16 h of serum starvation, cells were incubated with anti-EGFR antibodies (LA-22) for 2 h at 37°C, followed by goat anti-mouse IgM antibodies conjugated to 10 nm gold for 1 h at 37°C. After washing to remove antibodies from the medium, the cells were stimulated with EGF for 10 min and then fixed. The localization of EGFR was examined by silver-enhanced immunogold electron microscopy. Scale bar, 100 nm. (g) HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with EGFR. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Cell lysates were immunoblotted with indicated antibodies. Tubulin serves as a loading control.
Mentions: EGFR localized in clathrin-coated microdomains on endosomes is sorted into intraluminal vesicles (ILVs) within MVBs and delivered to lysosomes for degradation. We therefore examined whether LRRK1 knockdown affects the sorting of EGFR into ILVs of enlarged endosomes using three-dimensional reconstructions. In control siRNA-transfected cells, Alexa 647-EGF was translocated from the limiting membrane to ILVs of Rab5(Q79L)-induced enlarged endosomes at 30 min after EGF stimulation (Fig. 8a). In contrast, depletion of LRRK1 reduced the intraluminal transport of Alexa 647-EGF into enlarged endosomes (Fig. 8b). Line intensity profiles of individual endosomes clearly showed that most of the EGF was localized within the inner membranes in control cells (Fig. 8c), but were retained in the limiting membrane in LRRK1-depleted cells (Fig. 8d). Quantification of EGF labelling showed that about 66.2% of the labelling was localized to the inner membranes in cells treated with control siRNA, whereas only 16.7% showed this localization in cells treated with LRRK1 siRNA (Fig. 8e). These results demonstrate that LRRK1 is needed for sorting of EGF/EGFR into ILVs of MVBs. When siRNA-resistant, wild-type GFP-LRRK1 was expressed in LRRK1-depleted cells, the defect in the intraluminal transport of Alexa 647-EGF was partially rescued (Fig. 8e). However, expression of the siRNA-resistant, kinase-negative GFP-LRRK1(K1243M) was able to rescue the defect in EGF sorting in LRRK1-depleted cells (Fig. 8e), suggesting that LRRK1 kinase activity is not essential for this step. Thus, LRRK1 functions as a scaffold in this process, independent of its kinase activity.

Bottom Line: Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments.Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes.Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Graduate school of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

ABSTRACT
Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments. In this paper, we demonstrate that leucine-rich repeat kinase LRRK1, which contains a MAPKKK-like kinase domain, forms a complex with activated EGFR through an interaction with Grb2. Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes. LRRK1 regulates EGFR transport from early to late endosomes and regulates the motility of EGF-containing early endosomes in a manner dependent on its kinase activity. Furthermore, LRRK1 serves as a scaffold facilitating the interaction of EGFR with the endosomal sorting complex required for transport-0 complex, thus enabling efficient sorting of EGFR to the inner vesicles of multivesicular bodies. Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.

No MeSH data available.


Related in: MedlinePlus