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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 modulates the ubiquitination status of STAM1.(a) Effect of LRRK1 depletion on the ubiquitination of STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with Flag-STAM1 and HA-Ub, and stimulated with 100 ng per ml of EGF. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (b) Effect of LRRK1 overexpression on the ubiquitination of STAM1. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the K1243 mutant), Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (c) Ubiquitination of STAM1. COS7 cells were co-transfected with Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrowheads and lines indicate the positions of the monoubiquitinated and polyubiquitinated forms of Flag-STAM1, respectively. HC represents heavy chain. (d) Ubiquitination sites in STAM1. The lysine residues around the STAM1 coiled-coil domain (green) are shown. COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (e) Interaction between EGFR and STAM1(5KR). COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and EGFR as indicated, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies.
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f7: LRRK1 modulates the ubiquitination status of STAM1.(a) Effect of LRRK1 depletion on the ubiquitination of STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with Flag-STAM1 and HA-Ub, and stimulated with 100 ng per ml of EGF. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (b) Effect of LRRK1 overexpression on the ubiquitination of STAM1. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the K1243 mutant), Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (c) Ubiquitination of STAM1. COS7 cells were co-transfected with Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrowheads and lines indicate the positions of the monoubiquitinated and polyubiquitinated forms of Flag-STAM1, respectively. HC represents heavy chain. (d) Ubiquitination sites in STAM1. The lysine residues around the STAM1 coiled-coil domain (green) are shown. COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (e) Interaction between EGFR and STAM1(5KR). COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and EGFR as indicated, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies.

Mentions: The sorting of ubiquitinated EGFR is initiated by the recognition of EGFR ubiquitin (Ub) moieties by the Ub-interacting motifs (UIMs) within Hrs and STAM1032. Hrs and STAM themselves are also ubiquitinated3233, and a recent study has suggested that intramolecular binding between UIMs and Ub prevents Hrs from binding in trans to ubiquitinated EGFR32. We therefore examined the effect of LRRK1 knockdown on the ubiquitination status of STAM1. We co-transfected HeLa S3 cells with Flag-STAM1 and HA-Ub. In control siRNA cells, we could detect weak ubiquitination of STAM1 and this ubiquitination was enhanced by EGF stimulation (Fig. 7a). Depletion of LRRK1 resulted in hyper-ubiquitination of STAM1 (Fig. 7a). Thus, depletion of LRRK1 results in the enhancement of Ub modification in STAM1. We then examined the effect of LRRK1 overexpression on the ubiquitination status of STAM1. We found that overexpression of LRRK1 inhibited ubiquitination of STAM1 (Fig. 7b). In addition, overexpression of kinase-negative LRRK1(K1243M) was able to inhibit STAM1 ubiquitination (Fig. 7b), suggesting that LRRK1 kinase activity is not necessary for this reaction.


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 modulates the ubiquitination status of STAM1.(a) Effect of LRRK1 depletion on the ubiquitination of STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with Flag-STAM1 and HA-Ub, and stimulated with 100 ng per ml of EGF. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (b) Effect of LRRK1 overexpression on the ubiquitination of STAM1. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the K1243 mutant), Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (c) Ubiquitination of STAM1. COS7 cells were co-transfected with Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrowheads and lines indicate the positions of the monoubiquitinated and polyubiquitinated forms of Flag-STAM1, respectively. HC represents heavy chain. (d) Ubiquitination sites in STAM1. The lysine residues around the STAM1 coiled-coil domain (green) are shown. COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (e) Interaction between EGFR and STAM1(5KR). COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and EGFR as indicated, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies.
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f7: LRRK1 modulates the ubiquitination status of STAM1.(a) Effect of LRRK1 depletion on the ubiquitination of STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with Flag-STAM1 and HA-Ub, and stimulated with 100 ng per ml of EGF. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (b) Effect of LRRK1 overexpression on the ubiquitination of STAM1. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the K1243 mutant), Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibody were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (c) Ubiquitination of STAM1. COS7 cells were co-transfected with Flag-STAM1 and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrowheads and lines indicate the positions of the monoubiquitinated and polyubiquitinated forms of Flag-STAM1, respectively. HC represents heavy chain. (d) Ubiquitination sites in STAM1. The lysine residues around the STAM1 coiled-coil domain (green) are shown. COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and HA-Ub as indicated. Complexes immunoprecipitated (IP) with anti-Flag antibodies were immunoblotted (Blot) with anti-HA antibodies. Arrows and line indicate the positions of the unmodified and ubiquitinated forms of Flag-STAM1, respectively. (e) Interaction between EGFR and STAM1(5KR). COS7 cells were co-transfected with Flag-STAM1 (wild type and the 5KR mutant) and EGFR as indicated, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies.
Mentions: The sorting of ubiquitinated EGFR is initiated by the recognition of EGFR ubiquitin (Ub) moieties by the Ub-interacting motifs (UIMs) within Hrs and STAM1032. Hrs and STAM themselves are also ubiquitinated3233, and a recent study has suggested that intramolecular binding between UIMs and Ub prevents Hrs from binding in trans to ubiquitinated EGFR32. We therefore examined the effect of LRRK1 knockdown on the ubiquitination status of STAM1. We co-transfected HeLa S3 cells with Flag-STAM1 and HA-Ub. In control siRNA cells, we could detect weak ubiquitination of STAM1 and this ubiquitination was enhanced by EGF stimulation (Fig. 7a). Depletion of LRRK1 resulted in hyper-ubiquitination of STAM1 (Fig. 7a). Thus, depletion of LRRK1 results in the enhancement of Ub modification in STAM1. We then examined the effect of LRRK1 overexpression on the ubiquitination status of STAM1. We found that overexpression of LRRK1 inhibited ubiquitination of STAM1 (Fig. 7b). In addition, overexpression of kinase-negative LRRK1(K1243M) was able to inhibit STAM1 ubiquitination (Fig. 7b), suggesting that LRRK1 kinase activity is not necessary for this reaction.

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