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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

Localization of LRRK1 and EGFR on STAM1-positive microdomains of early endosomes.(a, b) Localization of LRRK1 and STAM1 on enlarged endosomes. HeLa S3 cells were co-transfected with DsRed-Rab5(Q79L) and GFP-LRRK1, and stimulated without (a) or with (b) 100 ng per ml of EGF for 10 min. The cells were fixed and stained with anti-STAM1 antibodies and imaged by confocal microscopy. Experiments were conducted three times with similar results. Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control (c) or LRRK1 siRNA (Stealth#1) (d) were transfected with GFP-Rab5(Q79L) and stimulated with 100 ng per ml of EGF for 10 min. The cells were fixed, stained with anti-EGFR and anti-STAM1 antibodies and imaged by confocal microscopy. The boxed regions indicate the Rab5(Q79L)-induced enlarged endosomes and are magnified in the inset. Scale bar, 10 μm. (e) Quantification of EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5(Q79L), and stimulated with 100 ng per ml of EGF for the indicated times. The cells were fixed, and stained with anti-EGFR and anti-STAM1 antibodies. Data show the percentage of STAM1-labelled EGFR-positive microdomains per endosomes. Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 80 endosomes) scored per experiment.
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f6: Localization of LRRK1 and EGFR on STAM1-positive microdomains of early endosomes.(a, b) Localization of LRRK1 and STAM1 on enlarged endosomes. HeLa S3 cells were co-transfected with DsRed-Rab5(Q79L) and GFP-LRRK1, and stimulated without (a) or with (b) 100 ng per ml of EGF for 10 min. The cells were fixed and stained with anti-STAM1 antibodies and imaged by confocal microscopy. Experiments were conducted three times with similar results. Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control (c) or LRRK1 siRNA (Stealth#1) (d) were transfected with GFP-Rab5(Q79L) and stimulated with 100 ng per ml of EGF for 10 min. The cells were fixed, stained with anti-EGFR and anti-STAM1 antibodies and imaged by confocal microscopy. The boxed regions indicate the Rab5(Q79L)-induced enlarged endosomes and are magnified in the inset. Scale bar, 10 μm. (e) Quantification of EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5(Q79L), and stimulated with 100 ng per ml of EGF for the indicated times. The cells were fixed, and stained with anti-EGFR and anti-STAM1 antibodies. Data show the percentage of STAM1-labelled EGFR-positive microdomains per endosomes. Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 80 endosomes) scored per experiment.

Mentions: To further evaluate the interaction between LRRK1 and STAM, we examined the potential co-localization of LRRK1 and STAM1 in early endosomes. Previous studies have revealed that Hrs accumulates in restricted microdomains on the endosome membrane, strongly co-localizing with clathrin26272829. By contrast, EEA1-containing regions of the endosome membrane are largely devoid of Hrs and clathrin. To study these endosomal microdomains, HeLa S3 cells were transfected with a constitutively active form of Rab5(Q79L), to increase the fusion rate of endosomes30. High Rab5 activity results in enlarged early endosomes on which microdomains can be easily distinguished by confocal immunofluorescence microscopy26272931. STAM1 co-localized strongly with clathrin on the enlarged endosomes (Supplementary Fig. S13a), whereas there was little co-localization of STAM1 and EEA1 (Supplementary Fig. S13b). These results suggest that STAM1 participates together with Hrs in the sorting of ubiquitinated cargo in clathrin-coated microdomains of early endosomes. GFP-LRRK1 was mainly distributed to the cytosol in the absence of EGF stimulation (Fig. 6a). However, LRRK1 localization was restricted to the enlarged endosomes of EGF-treated cells, and we could detect a certain degree of localization of GFP-LRRK1 to STAM1-positive microdomains (Fig. 6b). We also found that EGFR co-localized with STAM1 on Rab5(Q79L)-induced enlarged endosomes after EGF stimulation (Fig. 6c). To investigate whether LRRK1 is important for the recruitment of EGFR into STAM1-positive microdomains, we set out to study the relative localizations of EGFR and STAM1 in LRRK1-depleted cells. In such LRRK1-depleted cells, both EGFR and STAM1 could still be detected on endosomes. Interestingly, however, LRRK1-depleted endosomes appeared to show decreased co-localization of EGFR and STAM1 (Fig. 6d). Furthermore, this co-localization gradually increased at the later phase of EGF stimulation (Fig. 6e), indicating that LRRK1 depletion delays EGFR-STAM1 co-localization. We confirmed the effect of LRRK1 depletion on the co-localization of Alexa 647-EGF and STAM1 by three-dimensional reconstruction of confocal Z-stack images (Supplementary Fig. S14). These results suggest that LRRK1 determines the efficiency of recruitment by serving as a scaffolding function on the endosomal membrane to restrict EGFR localization to STAM-positive microdomains.


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)

Localization of LRRK1 and EGFR on STAM1-positive microdomains of early endosomes.(a, b) Localization of LRRK1 and STAM1 on enlarged endosomes. HeLa S3 cells were co-transfected with DsRed-Rab5(Q79L) and GFP-LRRK1, and stimulated without (a) or with (b) 100 ng per ml of EGF for 10 min. The cells were fixed and stained with anti-STAM1 antibodies and imaged by confocal microscopy. Experiments were conducted three times with similar results. Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control (c) or LRRK1 siRNA (Stealth#1) (d) were transfected with GFP-Rab5(Q79L) and stimulated with 100 ng per ml of EGF for 10 min. The cells were fixed, stained with anti-EGFR and anti-STAM1 antibodies and imaged by confocal microscopy. The boxed regions indicate the Rab5(Q79L)-induced enlarged endosomes and are magnified in the inset. Scale bar, 10 μm. (e) Quantification of EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5(Q79L), and stimulated with 100 ng per ml of EGF for the indicated times. The cells were fixed, and stained with anti-EGFR and anti-STAM1 antibodies. Data show the percentage of STAM1-labelled EGFR-positive microdomains per endosomes. Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 80 endosomes) scored per experiment.
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f6: Localization of LRRK1 and EGFR on STAM1-positive microdomains of early endosomes.(a, b) Localization of LRRK1 and STAM1 on enlarged endosomes. HeLa S3 cells were co-transfected with DsRed-Rab5(Q79L) and GFP-LRRK1, and stimulated without (a) or with (b) 100 ng per ml of EGF for 10 min. The cells were fixed and stained with anti-STAM1 antibodies and imaged by confocal microscopy. Experiments were conducted three times with similar results. Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control (c) or LRRK1 siRNA (Stealth#1) (d) were transfected with GFP-Rab5(Q79L) and stimulated with 100 ng per ml of EGF for 10 min. The cells were fixed, stained with anti-EGFR and anti-STAM1 antibodies and imaged by confocal microscopy. The boxed regions indicate the Rab5(Q79L)-induced enlarged endosomes and are magnified in the inset. Scale bar, 10 μm. (e) Quantification of EGFR localization to STAM-positive microdomains of early endosomes. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5(Q79L), and stimulated with 100 ng per ml of EGF for the indicated times. The cells were fixed, and stained with anti-EGFR and anti-STAM1 antibodies. Data show the percentage of STAM1-labelled EGFR-positive microdomains per endosomes. Values reflect the mean s.d. of three independent experiments, with an average of 15 cells (total 80 endosomes) scored per experiment.
Mentions: To further evaluate the interaction between LRRK1 and STAM, we examined the potential co-localization of LRRK1 and STAM1 in early endosomes. Previous studies have revealed that Hrs accumulates in restricted microdomains on the endosome membrane, strongly co-localizing with clathrin26272829. By contrast, EEA1-containing regions of the endosome membrane are largely devoid of Hrs and clathrin. To study these endosomal microdomains, HeLa S3 cells were transfected with a constitutively active form of Rab5(Q79L), to increase the fusion rate of endosomes30. High Rab5 activity results in enlarged early endosomes on which microdomains can be easily distinguished by confocal immunofluorescence microscopy26272931. STAM1 co-localized strongly with clathrin on the enlarged endosomes (Supplementary Fig. S13a), whereas there was little co-localization of STAM1 and EEA1 (Supplementary Fig. S13b). These results suggest that STAM1 participates together with Hrs in the sorting of ubiquitinated cargo in clathrin-coated microdomains of early endosomes. GFP-LRRK1 was mainly distributed to the cytosol in the absence of EGF stimulation (Fig. 6a). However, LRRK1 localization was restricted to the enlarged endosomes of EGF-treated cells, and we could detect a certain degree of localization of GFP-LRRK1 to STAM1-positive microdomains (Fig. 6b). We also found that EGFR co-localized with STAM1 on Rab5(Q79L)-induced enlarged endosomes after EGF stimulation (Fig. 6c). To investigate whether LRRK1 is important for the recruitment of EGFR into STAM1-positive microdomains, we set out to study the relative localizations of EGFR and STAM1 in LRRK1-depleted cells. In such LRRK1-depleted cells, both EGFR and STAM1 could still be detected on endosomes. Interestingly, however, LRRK1-depleted endosomes appeared to show decreased co-localization of EGFR and STAM1 (Fig. 6d). Furthermore, this co-localization gradually increased at the later phase of EGF stimulation (Fig. 6e), indicating that LRRK1 depletion delays EGFR-STAM1 co-localization. We confirmed the effect of LRRK1 depletion on the co-localization of Alexa 647-EGF and STAM1 by three-dimensional reconstruction of confocal Z-stack images (Supplementary Fig. S14). These results suggest that LRRK1 determines the efficiency of recruitment by serving as a scaffolding function on the endosomal membrane to restrict EGFR localization to STAM-positive microdomains.

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