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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 interacts with the ESCRT-0 complex in response to EGF stimulation.(a) Association of LRRK1 with STAM1. COS7 cells were transfected with GFP-LRRK1, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-STAM1 antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (b) Association of LRRK1 with Hrs. After 16 h of serum starvation, COS7 cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Hrs antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. (c) Schematic diagram of STAM1 protein. Deletion constructs of STAM1 are shown below. (d) Interaction of STAM1 fragments with LRRK1. COS7 cells were co-transfected with Flag-STAM1 and GFP-LRRK1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (e) Interaction of STAM1 with LRRK1 variants. COS7 cells were co-transfected with GFP-LRRK1 (wild type, K1243M and 4PA mutants) and Flag-STAM1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (f) Both wild-type LRRK1 and LRRK1(K1243M) form a ternary complex with EGFR and STAM1 in response to EGF stimulation. COS7 cells were co-transfected with Myc-LRRK1 (wild type and the K1243 mutant) and Flag-STAM1, as indicated, stimulated with 100 ng per ml of EGF, and collected for the first immunoprecipitation (IP) with anti-Flag antibody-conjugated agarose. Thereafter, the immunocomplexes were eluted by 3xFlag peptide and subjected to the second immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-Myc and anti-Flag antibodies. (g) LRRK1 is required for the interaction between EGFR and STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with EGFR and Flag-STAM1, 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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f5: LRRK1 interacts with the ESCRT-0 complex in response to EGF stimulation.(a) Association of LRRK1 with STAM1. COS7 cells were transfected with GFP-LRRK1, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-STAM1 antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (b) Association of LRRK1 with Hrs. After 16 h of serum starvation, COS7 cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Hrs antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. (c) Schematic diagram of STAM1 protein. Deletion constructs of STAM1 are shown below. (d) Interaction of STAM1 fragments with LRRK1. COS7 cells were co-transfected with Flag-STAM1 and GFP-LRRK1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (e) Interaction of STAM1 with LRRK1 variants. COS7 cells were co-transfected with GFP-LRRK1 (wild type, K1243M and 4PA mutants) and Flag-STAM1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (f) Both wild-type LRRK1 and LRRK1(K1243M) form a ternary complex with EGFR and STAM1 in response to EGF stimulation. COS7 cells were co-transfected with Myc-LRRK1 (wild type and the K1243 mutant) and Flag-STAM1, as indicated, stimulated with 100 ng per ml of EGF, and collected for the first immunoprecipitation (IP) with anti-Flag antibody-conjugated agarose. Thereafter, the immunocomplexes were eluted by 3xFlag peptide and subjected to the second immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-Myc and anti-Flag antibodies. (g) LRRK1 is required for the interaction between EGFR and STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with EGFR and Flag-STAM1, 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: Trafficking of EGF/EGFR in early endosomes is initiated by the interaction of ubiquitinated EGFR with components of the ESCRT-0 complex, STAM and Hrs678910. To determine whether the effect of LRRK1 on EGFR trafficking involves these endosomal sorting components, we examined the interaction of LRRK1 with STAM and Hrs. COS7 cells were transfected with GFP-LRRK1 and stimulated with EGF. Coimmunoprecipitation experiments revealed that LRRK1 associated with endogenous STAM1 and that this interaction was increased by EGF stimulation (Fig. 5a). Furthermore, we found that endogenous LRRK1 was coimmunoprecipitated with endogenous Hrs in an EGF stimulation-dependent manner (Fig. 5b). These results demonstrate that LRRK1 interacts with the ESCRT-0 complex in response to EGF stimulation.


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 interacts with the ESCRT-0 complex in response to EGF stimulation.(a) Association of LRRK1 with STAM1. COS7 cells were transfected with GFP-LRRK1, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-STAM1 antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (b) Association of LRRK1 with Hrs. After 16 h of serum starvation, COS7 cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Hrs antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. (c) Schematic diagram of STAM1 protein. Deletion constructs of STAM1 are shown below. (d) Interaction of STAM1 fragments with LRRK1. COS7 cells were co-transfected with Flag-STAM1 and GFP-LRRK1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (e) Interaction of STAM1 with LRRK1 variants. COS7 cells were co-transfected with GFP-LRRK1 (wild type, K1243M and 4PA mutants) and Flag-STAM1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (f) Both wild-type LRRK1 and LRRK1(K1243M) form a ternary complex with EGFR and STAM1 in response to EGF stimulation. COS7 cells were co-transfected with Myc-LRRK1 (wild type and the K1243 mutant) and Flag-STAM1, as indicated, stimulated with 100 ng per ml of EGF, and collected for the first immunoprecipitation (IP) with anti-Flag antibody-conjugated agarose. Thereafter, the immunocomplexes were eluted by 3xFlag peptide and subjected to the second immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-Myc and anti-Flag antibodies. (g) LRRK1 is required for the interaction between EGFR and STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with EGFR and Flag-STAM1, 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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f5: LRRK1 interacts with the ESCRT-0 complex in response to EGF stimulation.(a) Association of LRRK1 with STAM1. COS7 cells were transfected with GFP-LRRK1, and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-STAM1 antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (b) Association of LRRK1 with Hrs. After 16 h of serum starvation, COS7 cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-Hrs antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. (c) Schematic diagram of STAM1 protein. Deletion constructs of STAM1 are shown below. (d) Interaction of STAM1 fragments with LRRK1. COS7 cells were co-transfected with Flag-STAM1 and GFP-LRRK1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (e) Interaction of STAM1 with LRRK1 variants. COS7 cells were co-transfected with GFP-LRRK1 (wild type, K1243M and 4PA mutants) and Flag-STAM1, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (f) Both wild-type LRRK1 and LRRK1(K1243M) form a ternary complex with EGFR and STAM1 in response to EGF stimulation. COS7 cells were co-transfected with Myc-LRRK1 (wild type and the K1243 mutant) and Flag-STAM1, as indicated, stimulated with 100 ng per ml of EGF, and collected for the first immunoprecipitation (IP) with anti-Flag antibody-conjugated agarose. Thereafter, the immunocomplexes were eluted by 3xFlag peptide and subjected to the second immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-Myc and anti-Flag antibodies. (g) LRRK1 is required for the interaction between EGFR and STAM1. HeLa S3 cells treated with control or LRRK1 siRNA (Stealth#1) were co-transfected with EGFR and Flag-STAM1, 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: Trafficking of EGF/EGFR in early endosomes is initiated by the interaction of ubiquitinated EGFR with components of the ESCRT-0 complex, STAM and Hrs678910. To determine whether the effect of LRRK1 on EGFR trafficking involves these endosomal sorting components, we examined the interaction of LRRK1 with STAM and Hrs. COS7 cells were transfected with GFP-LRRK1 and stimulated with EGF. Coimmunoprecipitation experiments revealed that LRRK1 associated with endogenous STAM1 and that this interaction was increased by EGF stimulation (Fig. 5a). Furthermore, we found that endogenous LRRK1 was coimmunoprecipitated with endogenous Hrs in an EGF stimulation-dependent manner (Fig. 5b). These results demonstrate that LRRK1 interacts with the ESCRT-0 complex in response to EGF stimulation.

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