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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 forms a complex with EGFR via Grb2 in response to EGF stimulation.(a) Interaction of LRRK1 with Grb2. COS7 cells were co-transfected with Flag-LRRK1, Flag-LRRK2 and HA-Grb2 (wild type (WT), P49L, S90N, G203R and P49L/G203R mutants), as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-Flag antibodies. (b) Comparison of LRRK1 and LRRK2 structures. Schematic diagrams of human LRRK1 and LRRK2 proteins are shown. Domains are shown as follows: leucine-rich repeats (LRR), a Roc domain, a Cor domain, a MAPKKK-like kinase domain and WD40 repeats. The sequence alignment of LRRK1 and H-Ras in the Roc domain and of LRRK1 and mixed-lineage kinase 1 (MLK1) in the kinase domain are shown. Identical residues are highlighted with black shading. Arrowheads indicate amino-acid substitutions used in this study. Deletion constructs of LRRK1 are shown below. Two PXXP motifs within LRRK1(1–595) are shown by the arrowheads. (c) Effect of the 4PA mutation on the interaction of LRRK1 with Grb2. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the 4PA mutant) and HA-Grb2, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (d) Interaction of endogenous LRRK1 with endogenous Grb2. Complex formation in HEK293 cells was detected by immunoprecipitation (IP) with anti-Grb2 antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. LC, light chain. (e) Effect of the 4PA mutation on the interaction of LRRK1 with activated EGFR. COS7 cells were transfected with GFP-LRRK1 (wild type and the 4PA mutant). After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-GFP antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies. (f) Interaction of endogenous LRRK1 with endogenous EGFR. HEK293 cells treated with control or Grb2 siRNA were serum starved and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies.
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f1: LRRK1 forms a complex with EGFR via Grb2 in response to EGF stimulation.(a) Interaction of LRRK1 with Grb2. COS7 cells were co-transfected with Flag-LRRK1, Flag-LRRK2 and HA-Grb2 (wild type (WT), P49L, S90N, G203R and P49L/G203R mutants), as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-Flag antibodies. (b) Comparison of LRRK1 and LRRK2 structures. Schematic diagrams of human LRRK1 and LRRK2 proteins are shown. Domains are shown as follows: leucine-rich repeats (LRR), a Roc domain, a Cor domain, a MAPKKK-like kinase domain and WD40 repeats. The sequence alignment of LRRK1 and H-Ras in the Roc domain and of LRRK1 and mixed-lineage kinase 1 (MLK1) in the kinase domain are shown. Identical residues are highlighted with black shading. Arrowheads indicate amino-acid substitutions used in this study. Deletion constructs of LRRK1 are shown below. Two PXXP motifs within LRRK1(1–595) are shown by the arrowheads. (c) Effect of the 4PA mutation on the interaction of LRRK1 with Grb2. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the 4PA mutant) and HA-Grb2, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (d) Interaction of endogenous LRRK1 with endogenous Grb2. Complex formation in HEK293 cells was detected by immunoprecipitation (IP) with anti-Grb2 antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. LC, light chain. (e) Effect of the 4PA mutation on the interaction of LRRK1 with activated EGFR. COS7 cells were transfected with GFP-LRRK1 (wild type and the 4PA mutant). After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-GFP antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies. (f) Interaction of endogenous LRRK1 with endogenous EGFR. HEK293 cells treated with control or Grb2 siRNA were serum starved and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies.

Mentions: To investigate the physiological roles of LRRK1, we attempted to identify molecules with which it interacts. We used liquid chromatography-coupled tandem mass spectrometry (LC–MS/MS)17, and identified multiple peptides (Supplementary Table S1). Among the proteins identified, we focused on the Grb2 protein. To confirm the association between LRRK1 and Grb2, COS7 cells were co-transfected with Flag-LRRK1 and HA-Grb2. As seen in Figure 1a, Flag-LRRK1 associated with HA-Grb2. This association was specific for LRRK1, because LRRK2 did not interact with Grb2 (Fig. 1a).


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 forms a complex with EGFR via Grb2 in response to EGF stimulation.(a) Interaction of LRRK1 with Grb2. COS7 cells were co-transfected with Flag-LRRK1, Flag-LRRK2 and HA-Grb2 (wild type (WT), P49L, S90N, G203R and P49L/G203R mutants), as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-Flag antibodies. (b) Comparison of LRRK1 and LRRK2 structures. Schematic diagrams of human LRRK1 and LRRK2 proteins are shown. Domains are shown as follows: leucine-rich repeats (LRR), a Roc domain, a Cor domain, a MAPKKK-like kinase domain and WD40 repeats. The sequence alignment of LRRK1 and H-Ras in the Roc domain and of LRRK1 and mixed-lineage kinase 1 (MLK1) in the kinase domain are shown. Identical residues are highlighted with black shading. Arrowheads indicate amino-acid substitutions used in this study. Deletion constructs of LRRK1 are shown below. Two PXXP motifs within LRRK1(1–595) are shown by the arrowheads. (c) Effect of the 4PA mutation on the interaction of LRRK1 with Grb2. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the 4PA mutant) and HA-Grb2, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (d) Interaction of endogenous LRRK1 with endogenous Grb2. Complex formation in HEK293 cells was detected by immunoprecipitation (IP) with anti-Grb2 antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. LC, light chain. (e) Effect of the 4PA mutation on the interaction of LRRK1 with activated EGFR. COS7 cells were transfected with GFP-LRRK1 (wild type and the 4PA mutant). After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-GFP antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies. (f) Interaction of endogenous LRRK1 with endogenous EGFR. HEK293 cells treated with control or Grb2 siRNA were serum starved and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies.
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f1: LRRK1 forms a complex with EGFR via Grb2 in response to EGF stimulation.(a) Interaction of LRRK1 with Grb2. COS7 cells were co-transfected with Flag-LRRK1, Flag-LRRK2 and HA-Grb2 (wild type (WT), P49L, S90N, G203R and P49L/G203R mutants), as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-Flag antibodies. (b) Comparison of LRRK1 and LRRK2 structures. Schematic diagrams of human LRRK1 and LRRK2 proteins are shown. Domains are shown as follows: leucine-rich repeats (LRR), a Roc domain, a Cor domain, a MAPKKK-like kinase domain and WD40 repeats. The sequence alignment of LRRK1 and H-Ras in the Roc domain and of LRRK1 and mixed-lineage kinase 1 (MLK1) in the kinase domain are shown. Identical residues are highlighted with black shading. Arrowheads indicate amino-acid substitutions used in this study. Deletion constructs of LRRK1 are shown below. Two PXXP motifs within LRRK1(1–595) are shown by the arrowheads. (c) Effect of the 4PA mutation on the interaction of LRRK1 with Grb2. COS7 cells were co-transfected with GFP-LRRK1 (wild type and the 4PA mutant) and HA-Grb2, as indicated. Complex formation was detected by immunoprecipitation (IP) with anti-HA antibodies, followed by immunoblotting (Blot) with anti-GFP antibodies. (d) Interaction of endogenous LRRK1 with endogenous Grb2. Complex formation in HEK293 cells was detected by immunoprecipitation (IP) with anti-Grb2 antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies. LC, light chain. (e) Effect of the 4PA mutation on the interaction of LRRK1 with activated EGFR. COS7 cells were transfected with GFP-LRRK1 (wild type and the 4PA mutant). After 16 h of serum starvation, cells were stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-GFP antibodies, followed by immunoblotting (Blot) with anti-phospho-tyrosine (pTyr) antibodies. (f) Interaction of endogenous LRRK1 with endogenous EGFR. HEK293 cells treated with control or Grb2 siRNA were serum starved and stimulated with 100 ng per ml of EGF. Complex formation was detected by immunoprecipitation (IP) with anti-EGFR antibodies, followed by immunoblotting (Blot) with anti-LRRK1 antibodies.
Mentions: To investigate the physiological roles of LRRK1, we attempted to identify molecules with which it interacts. We used liquid chromatography-coupled tandem mass spectrometry (LC–MS/MS)17, and identified multiple peptides (Supplementary Table S1). Among the proteins identified, we focused on the Grb2 protein. To confirm the association between LRRK1 and Grb2, COS7 cells were co-transfected with Flag-LRRK1 and HA-Grb2. As seen in Figure 1a, Flag-LRRK1 associated with HA-Grb2. This association was specific for LRRK1, because LRRK2 did not interact with Grb2 (Fig. 1a).

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