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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

Depletion of LRRK1 inhibits EGFR transport from early to late endosomes.(a, b) HeLa S3 cells treated with control siRNA were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (a) and 15 min (b) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads and arrows indicate co-localization of Alexa 647-EGF with GFP-Rab5 and DsRed-Rab7, respectively (Supplementary Fig. S8). Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR transport from early to late endosomes. HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (c) and 15 min (d) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads indicate co-localization of Alexa 647-EGF with GFP-Rab5 (Supplementary Fig. S8). Scale bar, 10 μm. (e, f) Quantification of co-localization of EGFR with Rab5 (e) and Rab7 (f). Cells treated with control (blue) or LRRK1 siRNA (Stealth#1) (red) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min and 15 min after the initial exposure to Alexa 647-EGF. Data are presented as percentages of Rab5-labelled (e) or Rab7-labelled (f) Alexa647-EGF-containing vesicles out of the total number of Alexa647-EGF-containing vesicles per cell. Values reflect the mean s.d. of three independent experiments, with an average of ten cells scored per experiment.
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f3: Depletion of LRRK1 inhibits EGFR transport from early to late endosomes.(a, b) HeLa S3 cells treated with control siRNA were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (a) and 15 min (b) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads and arrows indicate co-localization of Alexa 647-EGF with GFP-Rab5 and DsRed-Rab7, respectively (Supplementary Fig. S8). Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR transport from early to late endosomes. HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (c) and 15 min (d) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads indicate co-localization of Alexa 647-EGF with GFP-Rab5 (Supplementary Fig. S8). Scale bar, 10 μm. (e, f) Quantification of co-localization of EGFR with Rab5 (e) and Rab7 (f). Cells treated with control (blue) or LRRK1 siRNA (Stealth#1) (red) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min and 15 min after the initial exposure to Alexa 647-EGF. Data are presented as percentages of Rab5-labelled (e) or Rab7-labelled (f) Alexa647-EGF-containing vesicles out of the total number of Alexa647-EGF-containing vesicles per cell. Values reflect the mean s.d. of three independent experiments, with an average of ten cells scored per experiment.

Mentions: Because it is known that Rab5 and Rab7 primarily associate with early and late endosomes, respectively, we coexpressed GFP-Rab5 and DsRed-Rab7 in HeLa S3 cells to visualize early and late endosomes. In this experiment, we used the monomer version of DsRed. Most of the Rab5-positive endosomes were distributed in the cell periphery, whereas Rab7-positive endosomes were more common in the perinuclear region (Fig. 3a–d). In control siRNA-transfected cells, at 10 min after a brief pulse of Alexa 647-EGF stimulation, most of the EGF was found in early endosomes, as evidenced by their co-localization with Rab5 (Fig. 3a,e; Supplementary Fig. S8a). After 15 min, EGF dissociated from the early endosomes, as illustrated by decreased co-localization with Rab5. Remarkably, this decreased co-localization with Rab5 was accompanied by an increase in the co-localization with Rab7 (Fig. 3b,e and f; Supplementary Fig. S8b), suggesting the progression of EGFR to the late-endosomal compartments. In LRRK1-depleted cells, the early endosomal localization of EGF occurred normally, as in control cells (Fig. 3c,e; Supplementary Fig. S8c). However, after 15 min of stimulation, Alexa 647-EGF remained associated with Rab5-positive structures and Rab7 co-localization did not increase (Fig. 3d–f; Supplementary Fig. S8d). This suggests that the transport of EGFR towards late endosomes is inhibited in LRRK1-deficient cells. Thus, LRRK1 is required for the progression of EGFR from early to late endosomes.


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)

Depletion of LRRK1 inhibits EGFR transport from early to late endosomes.(a, b) HeLa S3 cells treated with control siRNA were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (a) and 15 min (b) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads and arrows indicate co-localization of Alexa 647-EGF with GFP-Rab5 and DsRed-Rab7, respectively (Supplementary Fig. S8). Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR transport from early to late endosomes. HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (c) and 15 min (d) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads indicate co-localization of Alexa 647-EGF with GFP-Rab5 (Supplementary Fig. S8). Scale bar, 10 μm. (e, f) Quantification of co-localization of EGFR with Rab5 (e) and Rab7 (f). Cells treated with control (blue) or LRRK1 siRNA (Stealth#1) (red) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min and 15 min after the initial exposure to Alexa 647-EGF. Data are presented as percentages of Rab5-labelled (e) or Rab7-labelled (f) Alexa647-EGF-containing vesicles out of the total number of Alexa647-EGF-containing vesicles per cell. Values reflect the mean s.d. of three independent experiments, with an average of ten cells scored per experiment.
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f3: Depletion of LRRK1 inhibits EGFR transport from early to late endosomes.(a, b) HeLa S3 cells treated with control siRNA were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (a) and 15 min (b) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads and arrows indicate co-localization of Alexa 647-EGF with GFP-Rab5 and DsRed-Rab7, respectively (Supplementary Fig. S8). Scale bar, 10 μm. (c, d) Effect of LRRK1 depletion on EGFR transport from early to late endosomes. HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min (c) and 15 min (d) after the initial exposure to Alexa 647-EGF and imaged by confocal microscopy. The boxed regions are magnified in insets. Arrowheads indicate co-localization of Alexa 647-EGF with GFP-Rab5 (Supplementary Fig. S8). Scale bar, 10 μm. (e, f) Quantification of co-localization of EGFR with Rab5 (e) and Rab7 (f). Cells treated with control (blue) or LRRK1 siRNA (Stealth#1) (red) were co-transfected with GFP-Rab5 and DsRed-Rab7. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of Alexa 647-EGF for 3 min at 37°C, followed by washing to remove labelled EGF from the medium. The cells were fixed at 10 min and 15 min after the initial exposure to Alexa 647-EGF. Data are presented as percentages of Rab5-labelled (e) or Rab7-labelled (f) Alexa647-EGF-containing vesicles out of the total number of Alexa647-EGF-containing vesicles per cell. Values reflect the mean s.d. of three independent experiments, with an average of ten cells scored per experiment.
Mentions: Because it is known that Rab5 and Rab7 primarily associate with early and late endosomes, respectively, we coexpressed GFP-Rab5 and DsRed-Rab7 in HeLa S3 cells to visualize early and late endosomes. In this experiment, we used the monomer version of DsRed. Most of the Rab5-positive endosomes were distributed in the cell periphery, whereas Rab7-positive endosomes were more common in the perinuclear region (Fig. 3a–d). In control siRNA-transfected cells, at 10 min after a brief pulse of Alexa 647-EGF stimulation, most of the EGF was found in early endosomes, as evidenced by their co-localization with Rab5 (Fig. 3a,e; Supplementary Fig. S8a). After 15 min, EGF dissociated from the early endosomes, as illustrated by decreased co-localization with Rab5. Remarkably, this decreased co-localization with Rab5 was accompanied by an increase in the co-localization with Rab7 (Fig. 3b,e and f; Supplementary Fig. S8b), suggesting the progression of EGFR to the late-endosomal compartments. In LRRK1-depleted cells, the early endosomal localization of EGF occurred normally, as in control cells (Fig. 3c,e; Supplementary Fig. S8c). However, after 15 min of stimulation, Alexa 647-EGF remained associated with Rab5-positive structures and Rab7 co-localization did not increase (Fig. 3d–f; Supplementary Fig. S8d). This suggests that the transport of EGFR towards late endosomes is inhibited in LRRK1-deficient cells. Thus, LRRK1 is required for the progression of EGFR from early to late endosomes.

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