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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 the motility of EGF-containing early endosomes.(a) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with control siRNA were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (b) Stills from control cells (the boxed region in a). Arrowheads and arrows indicate the movement of GFP-Rab5-positive endosome containing Alexa 647-EGF. (c) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (d) Stills from LRRK1-depleted cells (the boxed region in c). (e) A mobility histogram of randomly selected Rab5- and EGF-double-positive endosomes from control (blue) or LRRK1-depleted cells (red). Mobility is defined as the distance of the trajectory of endosomes during the 35 s observation period, quantified using the 'Manual Tracking' Image J plug-in. Values reflect the mean s.d. of five independent experiments, with an average of 30 Rab5- and EGF-double-positive endosomes scored per experiment.
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f4: Depletion of LRRK1 inhibits the motility of EGF-containing early endosomes.(a) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with control siRNA were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (b) Stills from control cells (the boxed region in a). Arrowheads and arrows indicate the movement of GFP-Rab5-positive endosome containing Alexa 647-EGF. (c) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (d) Stills from LRRK1-depleted cells (the boxed region in c). (e) A mobility histogram of randomly selected Rab5- and EGF-double-positive endosomes from control (blue) or LRRK1-depleted cells (red). Mobility is defined as the distance of the trajectory of endosomes during the 35 s observation period, quantified using the 'Manual Tracking' Image J plug-in. Values reflect the mean s.d. of five independent experiments, with an average of 30 Rab5- and EGF-double-positive endosomes scored per experiment.

Mentions: Because maturation of early to late endosomes is accompanied by the movement of endosomes towards the cell centre181920, we examined the effect of LRRK1 knockdown on the movement of EGF-containing early endosomes. Using time-lapse confocal fluorescence microscopy, movement of EGF-containing early endosomes was followed in cells expressing GFP-Rab5 at 15 min after a brief pulse of Alexa647-EGF. In addition to short-range movement, long-range rapid movement of EGF-positive early endosomes from the periphery to the centre was frequently observed (Fig. 4a,b). When LRRK1 was knocked down by siRNA, long-range rapid movement of EGF-containing early endosomes was remarkably disrupted (Fig. 4c,d). In control cells, 39.7% of GFP-Rab5- and Alexa 647-EGF-double-positive endosomes moved more than 3.0 μm during the imaging time (35 s), but this fraction decreased to only 2.4% in LRRK1-depleted cells (Fig. 4e). We further confirmed this effect of knocking down LRRK1 on the frequency of long-range movement of EGF using a different LRRK1 siRNA in different HeLa cell lines (Supplementary Fig. S9). These observations suggest that LRRK1 is required for the motility of EGF-containing early 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 the motility of EGF-containing early endosomes.(a) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with control siRNA were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (b) Stills from control cells (the boxed region in a). Arrowheads and arrows indicate the movement of GFP-Rab5-positive endosome containing Alexa 647-EGF. (c) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (d) Stills from LRRK1-depleted cells (the boxed region in c). (e) A mobility histogram of randomly selected Rab5- and EGF-double-positive endosomes from control (blue) or LRRK1-depleted cells (red). Mobility is defined as the distance of the trajectory of endosomes during the 35 s observation period, quantified using the 'Manual Tracking' Image J plug-in. Values reflect the mean s.d. of five independent experiments, with an average of 30 Rab5- and EGF-double-positive endosomes scored per experiment.
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f4: Depletion of LRRK1 inhibits the motility of EGF-containing early endosomes.(a) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with control siRNA were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (b) Stills from control cells (the boxed region in a). Arrowheads and arrows indicate the movement of GFP-Rab5-positive endosome containing Alexa 647-EGF. (c) Images representing GFP-Rab5-positive endosomes containing Alexa 647-EGF (red circles) and tracks (yellow). HeLa S3 cells treated with LRRK1 siRNA (Stealth#1) were transfected with GFP-Rab5, pulse-labelled with Alexa 647-EGF. Movement of GFP-Rab5-positive endosomes containing Alexa 647-EGF was observed by time-lapse confocal fluorescence microscopy and analysed using the 'Manual Tracking' Image J plug-in. Imaging started at 15 min after the initial exposure to Alexa 647-EGF, with frames captured at 1.165 s intervals for 35 s. Scale bar, 10 μm. (d) Stills from LRRK1-depleted cells (the boxed region in c). (e) A mobility histogram of randomly selected Rab5- and EGF-double-positive endosomes from control (blue) or LRRK1-depleted cells (red). Mobility is defined as the distance of the trajectory of endosomes during the 35 s observation period, quantified using the 'Manual Tracking' Image J plug-in. Values reflect the mean s.d. of five independent experiments, with an average of 30 Rab5- and EGF-double-positive endosomes scored per experiment.
Mentions: Because maturation of early to late endosomes is accompanied by the movement of endosomes towards the cell centre181920, we examined the effect of LRRK1 knockdown on the movement of EGF-containing early endosomes. Using time-lapse confocal fluorescence microscopy, movement of EGF-containing early endosomes was followed in cells expressing GFP-Rab5 at 15 min after a brief pulse of Alexa647-EGF. In addition to short-range movement, long-range rapid movement of EGF-positive early endosomes from the periphery to the centre was frequently observed (Fig. 4a,b). When LRRK1 was knocked down by siRNA, long-range rapid movement of EGF-containing early endosomes was remarkably disrupted (Fig. 4c,d). In control cells, 39.7% of GFP-Rab5- and Alexa 647-EGF-double-positive endosomes moved more than 3.0 μm during the imaging time (35 s), but this fraction decreased to only 2.4% in LRRK1-depleted cells (Fig. 4e). We further confirmed this effect of knocking down LRRK1 on the frequency of long-range movement of EGF using a different LRRK1 siRNA in different HeLa cell lines (Supplementary Fig. S9). These observations suggest that LRRK1 is required for the motility of EGF-containing early 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