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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

LRRK1 co-localizes with EGF in early endosomes.(a,b) Co-localization of LRRK1 with EGF in EEA1-labelled early endosomes. HeLa cells were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were incubated without (a) or with (b) 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. The boxed regions are magnified in insets. Examples of co-localization of GFP-LRRK1, EGF-Rh and EEA1 are indicated with arrowheads (Supplementary Fig. S4). Scale bar, 10 μm. (c, d) Effect of Grb2 depletion on LRRK1 localization in early endosomes. HeLa S3 cells treated with control (c) or Grb2 siRNA (d) were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF for 15 min. The cells were fixed, stained with anti-EEA1 antibodies and imaged by confocal microscopy. Yellow colour in the merged images indicates co-localization. Scale bar, 10 μm. (e) Effect of 4PA mutation on LRRK1 localization. HeLa cells were transfected with GFP-LRRK1(4PA). After 16 h of serum starvation, cells were incubated with 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. Scale bar, 10 μm.
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f2: LRRK1 co-localizes with EGF in early endosomes.(a,b) Co-localization of LRRK1 with EGF in EEA1-labelled early endosomes. HeLa cells were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were incubated without (a) or with (b) 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. The boxed regions are magnified in insets. Examples of co-localization of GFP-LRRK1, EGF-Rh and EEA1 are indicated with arrowheads (Supplementary Fig. S4). Scale bar, 10 μm. (c, d) Effect of Grb2 depletion on LRRK1 localization in early endosomes. HeLa S3 cells treated with control (c) or Grb2 siRNA (d) were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF for 15 min. The cells were fixed, stained with anti-EEA1 antibodies and imaged by confocal microscopy. Yellow colour in the merged images indicates co-localization. Scale bar, 10 μm. (e) Effect of 4PA mutation on LRRK1 localization. HeLa cells were transfected with GFP-LRRK1(4PA). After 16 h of serum starvation, cells were incubated with 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. Scale bar, 10 μm.

Mentions: To investigate the role of LRRK1 in EGFR signalling, we examined the subcellular localization of LRRK1. Because our polyclonal anti-LRRK1 antibody did not provide a sensitive enough detection of the endogenous protein by immunostaining, we used an epitope-tagged LRRK1. We expressed GFP-LRRK1 in HeLa cells and assessed LRRK1 localization by immunofluorescence under basal and EGF-stimulated conditions, together with a fluorescently labelled rhodamine-conjugated EGF (EGF-Rh). LRRK1 was diffusely distributed in cells not treated with EGF-Rh (Fig. 2a). When cells were stimulated with EGF-Rh for 15 min, GFP-LRRK1 became distributed in a fine, punctate pattern that co-localized with EGF-Rh. A large portion of GFP-LRRK1 overlapped with the early endosomal marker autoantigen 1 (EEA1) receptor (Fig. 2b; Supplementary Fig. S4).


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)

LRRK1 co-localizes with EGF in early endosomes.(a,b) Co-localization of LRRK1 with EGF in EEA1-labelled early endosomes. HeLa cells were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were incubated without (a) or with (b) 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. The boxed regions are magnified in insets. Examples of co-localization of GFP-LRRK1, EGF-Rh and EEA1 are indicated with arrowheads (Supplementary Fig. S4). Scale bar, 10 μm. (c, d) Effect of Grb2 depletion on LRRK1 localization in early endosomes. HeLa S3 cells treated with control (c) or Grb2 siRNA (d) were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF for 15 min. The cells were fixed, stained with anti-EEA1 antibodies and imaged by confocal microscopy. Yellow colour in the merged images indicates co-localization. Scale bar, 10 μm. (e) Effect of 4PA mutation on LRRK1 localization. HeLa cells were transfected with GFP-LRRK1(4PA). After 16 h of serum starvation, cells were incubated with 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. Scale bar, 10 μm.
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f2: LRRK1 co-localizes with EGF in early endosomes.(a,b) Co-localization of LRRK1 with EGF in EEA1-labelled early endosomes. HeLa cells were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were incubated without (a) or with (b) 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. The boxed regions are magnified in insets. Examples of co-localization of GFP-LRRK1, EGF-Rh and EEA1 are indicated with arrowheads (Supplementary Fig. S4). Scale bar, 10 μm. (c, d) Effect of Grb2 depletion on LRRK1 localization in early endosomes. HeLa S3 cells treated with control (c) or Grb2 siRNA (d) were transfected with GFP-LRRK1. After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF for 15 min. The cells were fixed, stained with anti-EEA1 antibodies and imaged by confocal microscopy. Yellow colour in the merged images indicates co-localization. Scale bar, 10 μm. (e) Effect of 4PA mutation on LRRK1 localization. HeLa cells were transfected with GFP-LRRK1(4PA). After 16 h of serum starvation, cells were incubated with 50 ng per ml of EGF-Rh (red) for 45 min at 4°C, followed by warming for 15 min at 37°C, and stained with anti-EEA1 antibodies (blue). All cells were imaged by confocal microscopy. Scale bar, 10 μm.
Mentions: To investigate the role of LRRK1 in EGFR signalling, we examined the subcellular localization of LRRK1. Because our polyclonal anti-LRRK1 antibody did not provide a sensitive enough detection of the endogenous protein by immunostaining, we used an epitope-tagged LRRK1. We expressed GFP-LRRK1 in HeLa cells and assessed LRRK1 localization by immunofluorescence under basal and EGF-stimulated conditions, together with a fluorescently labelled rhodamine-conjugated EGF (EGF-Rh). LRRK1 was diffusely distributed in cells not treated with EGF-Rh (Fig. 2a). When cells were stimulated with EGF-Rh for 15 min, GFP-LRRK1 became distributed in a fine, punctate pattern that co-localized with EGF-Rh. A large portion of GFP-LRRK1 overlapped with the early endosomal marker autoantigen 1 (EEA1) receptor (Fig. 2b; Supplementary Fig. S4).

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