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1.
FIGURE 7.

FIGURE 7. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Eps15 knockdown is rescued by WT and P770A mutant but not rescued by Eps15ΔCC or Eps15ΔUIM mutants. A, HeLa cells treated with siRNA as described under “Experimental Procedures,” were transfected with either vector, siRNA-resistant Eps15 WT, Eps15ΔCC, Eps15 P770A, or Eps15ΔUIM. 24 h later, cells were treated with HGF in the presence of cycloheximide for 2 h, fixed, stained for Met and Eps15 (or vector), and viewed using confocal microscopy. Magnification, 100×; zoom, 1.5; bar = 10 μm. B, Eps15 knockdown cells expressing Eps15 constructs from A were counted for anti-Met staining. Results shown are from three independent experiments with at least 30 cells counted per condition. C, Western blot of knockdown cells expressing Eps15 constructs. Cells were stimulated with HGF for 2 h in the presence of cycloheximide, and protein levels were blotted as indicated.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.
2.
FIGURE 3.

FIGURE 3. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Eps15 is recruited to a Met complex through its coiled-coil domain. A, HEK293 cells were transiently transfected with Met and truncation mutants of Eps15, followed by immunoprecipitation of Met and FLAG-Eps15 48 h post-transfection and Western blotted (WB) as shown. B, individual domains of Eps15 II, III, or II and III were co-transfected with Met and immunoprecipitated as in A. Eps15 WT, Eps15ΔII and Eps15ΔIII were run out on the same gel for level comparison. C, schematic diagram of Eps15 mutants and a summary of the immunoprecipitation (IP) analysis. Binding to Met was characterized as strong (+++), good (++), weak (–/+) or poorly/not at all (–). D, FLAG-Eps15 and Met WT or Met Y1003F were transiently transfected in HEK293 cells. Lysates were immunoprecipitated for Met and FLAG then Western blotted as indicated. E, HEK293 cells were transiently co-transfected with EGFR and truncation mutants of Eps15. 24 h later cells were serum-starved overnight, then stimulated with 100 ng/ml EGF for 8 min, followed by immunoprecipitation of EGFR, and Western blotted as shown.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.
3.
FIGURE 2.

FIGURE 2. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Eps15 is subject to post-translational modifications in response to HGF stimulation. A, HeLa and T47D stables expressing Met were blotted for protein levels. B, T47D cells stably expressing either Met WT or Met K1110A (kinase-dead) were cold load stimulated with HGF for 1 h at 4 °C then released to 37 °C for the time indicated. Endogenous Eps15 or Met was immunoprecipitated (IP) and blotted for 4G10 anti-phosphotyrosine (pTyr) and total protein levels. AP2, mu2 subunit was also blotted. C, HeLa cells were cold load-stimulated, immunoprecipitated for endogenous Eps15, and blotted for tyrosine phosphorylation. D, HeLa cells were stimulated with HGF at 37 °C for the indicated times, and lysed. Endogenous Eps15 was immunoprecipitated and phosphotyrosine levels were assessed. E, HEK293 cells were co-transfected with GFP-Eps15 and increasing amounts of Met WT cDNA expression vector. Eps15 was immunoprecipitated using anti-GFP antibodies, and phosphotyrosine levels were examined. F, HEK293 cells were transfected with either FLAG-Eps15 WT or Y850F and co-transfected with Met WT or K1110A. Immunoprecipitations were performed using anti-Met or anti-FLAG M2 beads and analyzed through Western blotting as indicated. G, HeLa cells transfected with HA-Ubiquitin (HA-Ub) were cold load-stimulated with HGF and immunoprecipitated for Eps15 then blotted for HA-Ubiquitin, and reprobed for Eps15. UT = untransfected.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.
4.
FIGURE 4.

FIGURE 4. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Grb2 associates with Eps15 through a proline-rich motif. A, schematic of Eps15 and Grb2 domains. Proline 770 of Eps15 within the PXXP motif with surrounding amino acid identified using Scansite are indicated along with the score. The scores for one of the proline residues of SOS and Gab1 as identified by Scansite are also given for comparison. B, FLAG-Eps15 and Myc-Grb2 expression constructs were transfected with or without Met and immunoprecipitated (IP) as indicated followed by blotting for Eps15 (FLAG), Grb2 (myc), or Met levels. C, HeLa cells left unstimulated or stimulated with HGF for 15 min were lysed and immunoprecipitated using Eps15 antibodies (+Ab) and blotted for Grb2. Parallel control immunoprecipitates were carried out without antibody addition (–Ab). D, a panel of FLAG-Eps15 truncation mutants were co-transfected with myc-Grb2 in HEK293 cells and immunoprecipitated and blotted as indicated. E, summary of co-immunoprecipitations of Eps15 and Grb2, (+) = co-immunoprecipitation observed, (–) = no co-immunoprecipitation observed. F, GST pulldown assays using GST-Grb2 using lysates transfected with Eps15 WT or Eps15 P770A expression constructs. GST-alone was used as a control. G, HeLa cells transfected with Eps15 WT or P770A and HA-Ub were cold load-stimulated with 0.60 nm HGF and immunoprecipitated for Eps15 then blotted for HA and phosphotyrosine. Met activation was assessed through Western blotting using phosphospecific Met tyrosine 1234/35 antibodies.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.
5.
FIGURE 5.

FIGURE 5. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Coiled-coil domain is sufficient for displacing a WT-Eps15-Met complex but not an EGFR-Eps15 complex. A, HEK293 cells were transfected with Met and Eps15 WT expression constructs in the presence of increasing amounts of the coiled-coil domain (Eps15 CC). Immunoprecipitations (IP) were performed on the lysates as indicated. Arrows show migration of wild-type (Eps15 WT) and Eps15 CC proteins. Molecular weight protein marker migration is indicated to the left of the blots. B, HEK293 cells transfected with either Met or EGFR and FLAG-Eps15 expression constructs with increasing amounts of FLAG-CC. EGFR cells were stimulated with 100 ng/ml EGF for 8 min prior to lysing. Lysates were immunoprecipitated with anti-Met and anti-EGFR antibodies and blotted for FLAG. Arrows show migration of wild-type (Eps15 WT) and Eps15 CC proteins and heavy chain. Molecular weight protein marker migration is indicated to the left of the blots. C, quantification of coiled-coil protein levels co-immunoprecipitated with anti-Met or anti-EGFR antibodies with increasing CC domain protein levels. Results were quantified using densitometric analysis from at least three independent experiments. For Met immunoprecipitations; p < 0.03, Student's t test, mean ± S.E. D, quantification of FLAG-Eps15 WT protein levels co-immunoprecipitated in the presence of increasing amounts of coiled-coil domain using anti-Met and anti-EGFR antibodies. Results are quantified using densitometric analysis from at least three independent experiments. Shown is the mean ± S.E. For Met co-immunoprecipitation; p < 0.003, Student's t test.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.
6.
FIGURE 1.

FIGURE 1. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Eps15 is recruited to the Met receptor upon Met activation. A, schematic diagram depicting the experimental protocol for a cold load stimulation, used to assess synchronized internalization and trafficking of RTKs. B, confocal images of endogenous Eps15 and Met upon cold load stimulation. HeLa cells were serum-starved in the presence of cycloheximide to reduce newly synthesized Met staining, then fixed and imaged (a–c) or treated with HGF (0.60 nm) for 1 h at 4 °C (d–f), followed by warming to 37 °C for the indicated times (g–o). Met (red), Eps15 (green), and 4′,6-diamidino-2-phenylindole. Magnification, 100×; zoom, 1.5×; bar = 10 μm. C, HeLa cells were subjected to a cold load stimulation, and endogenous Eps15 was immunoprecipitated (1 mg) and blotted for Met and total Eps15 levels. Whole cell lysates (WCL) were blotted (WB) for phospho-Met (p1234/1235) and actin levels. D, HeLa cells were plated on coverslips. 24 h later, cells were serum-starved in the presence of cycloheximide for 2 h, followed by cold load stimulating with 0.12 nm HGF followed by warming to 37 °C for 15 min. Cells were stained for Met (red), Eps15 (green), and 4′,6-diamidino-2-phenylindole. Bar = 10 μm.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.
7.
FIGURE 6.

FIGURE 6. From: Distinct Recruitment of Eps15 via Its Coiled-coil Domain Is Required For Efficient Down-regulation of the Met Receptor Tyrosine Kinase.

Eps15 knockdown delays Met degradation and is rescued by WT Eps15. A, HeLa cells transfected with control scramble siRNA (10 nm) or Eps15 siRNA at 5 nm or 10 nm concentrations were harvested 72 h post-transfection and assessed for protein levels. B, HeLa cells transfected with scramble or Eps15 siRNA were stimulated with HGF in the presence of cycloheximide then stained for Met and 4′,6-diamidino-2-phenylindole and imaged using confocal microscopy for the indicated times. Arrows point to Met-positive staining. Bar = 20 μm. C, Western blots of HeLa cells transfected with scramble and Eps15 siRNA. Cells were stimulated with HGF in the presence of cycloheximide for the indicated amount of time. Lysates were blotted for Met, Eps15, and actin (top panel). Mean Met protein levels were quantified through densitometric analysis and graphed as percentage of initial protein levels. Error bars represent S.E. Results shown are from four independent experiments (bottom panel). D (left panel): HeLa cells transfected with Eps15 siRNA were transiently transfected with siRNA-resistant Eps15 cDNA and compared with scramble control or Eps15 siRNA-treated cells. Met protein levels were quantified as in C, after HGF stimulation in the presence of cycloheximide. Shown is the mean value of Met levels at the 4-h time point. Results are from four independent experiments. The difference between Eps15-depleted cells versus rescued cells is statistically significant; p < 0.01, Student's t test. Right panel: Western blots of siRNA-treated cells; *, GFP-Eps15 protein levels.

Christine A. Parachoniak, et al. J Biol Chem. 2009 March 27;284(13):8382-8394.

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