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First published online 2 December 2008
doi: 10.1242/jcs.037226

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A new mechanism for the regulation of Gab1 recruitment to the plasma membrane

Department of Biochemistry, RWTH Aachen University, Aachen, Germany

View larger version (30K): [in this window] [in a new window]   Fig. 1. The Grb2 and SHP2 binding sites in Gab1 are crucial for ERK activation. (A) HEK293T cells were cotransfected with expression vectors for Gab1, Gab1SHP2 or empty vector together with vectors encoding EpoR-gp130 chimeric receptors. Two days after transfection, cells were starved in DMEM without FCS and subsequently stimulated with Epo (7 U/ml) for 15 minutes to induce IL-6 signalling. Cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 (upper panel) to control stimulation of the cells, and for the activated forms of ERK1/2 (second panel). After stripping, the membrane was stained for exogenous Gab1 (Flag) and ERK to monitor Gab1-transfection efficiency and equal loading of the gel (ERK). (B) HEK293T cells were transfected with expression vectors encoding EpoR-gp130 together with vectors for wt-Gab1, Gab1SHP2 or empty vector. MAPK activity was monitored by using an Elk1 transactivation approach (PathDetect Elk1 trans Reporting System, Stratagene). For the Elk1 reporting system, cells were transfected with a Gal4-driven luciferase reporter and an expression vector encoding a fusion protein composed of the DNA-binding domain of Gal4 and the transactivation domain of the Elk1 transcription factor (pFA2-Elk1). Activation of MAPK leads to phosphorylation and activation of the Elk-transactivation domain and subsequent increase of Gal4/Elk1-dependent luciferase reporter activity. Modulation of MAPK activity by Gab1 mutants was monitored by luciferase activity. 6 hours after transfection, cells were stimulated with Epo (7 U/ml) for 16 hours to induce IL-6 signalling. Transfection efficiency was controlled by cotransfection of vector for constitutive expression of β-galactosidase. Luciferase activity was normalised to β-galactosidase activity of each sample in triplicate. Results are given as the mean ± s.e.m.

View larger version (48K): [in this window] [in a new window]   Fig. 2. The PH domain in Gab1 and PI3K activity are required for Gab1 activation. (A) HEK293T cells were cotransfected with expression vectors for wt-Gab1, Gab1PH or the isolated PH domain, together with vectors encoding EpoR-gp130 chimeric receptors. Two days after transfection, cells were starved in DMEM without FCS for 3 hours and subsequently stimulated with Epo (7 U/ml) for 15 minutes. Cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 to control stimulation of the cells and for the activated forms of ERK1/2 and Gab1. After stripping, the membrane was stained for Gab1 (Flag), STAT3 and ERK to monitor Gab1-transfection efficiency and equal loading of the gels (STAT3, ERK). (B) HEK293T cells were cotransfected with expression vectors for Gab1 and EpoR-gp130 chimeric receptors. Cells were treated as above and then with the PI3K inhibitor LY294002 (40 µM) for 45 minutes prior to stimulation with Epo (7 U/ml) for 15 minutes. After western blotting, the membranes were stained for Gab1 phosphorylated at Y627 and for Akt phosphorylated at Ser473 (pS473Akt) to monitor PI3K activity. After stripping, the membrane was stained for Gab1 and Akt to monitor Gab1-transfection efficiency and equal loading of the gel. The shift in Akt mobility is due to phosphorylation of the protein. Owing to the use of Gab1-GFP fusion proteins in the following experiments and to better distinguish endogenous from exogenous Gab1 proteins, the latter were expressed as GFP-fusion proteins to reduce mobility in SDS-PAGE.

View larger version (43K): [in this window] [in a new window]   Fig. 3. IL-6-induced translocation of Gab1 to the plasma membrane depends on basal PI3K activity. (A) HEK293T cells were transfected with expression vectors for Gab1-GFP together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes, the cells were stimulated with Epo (7 U/ml). Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation. (B) HEK293 and HEK293T cells were cotransfected with expression vectors for Gab1 and vectors encoding EpoR-gp130 chimeric receptors. Two days after transfection, cells were starved in DMEM without FCS for 3 hours and subsequently stimulated with Epo (7 U/ml) for 15 minutes. Cell lysates were prepared and equal amounts of protein separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 to control stimulation of the cells and for Akt phosphorylated at Ser473 (pS473Akt) to monitor PI3K activity. After stripping, the membrane was stained for STAT3 and AKT to monitor equal loading of the gel. (C,D) HEK293 and HEK293T cells were treated and Gab1 localisation analysed as described above in A (C) or pretreated with the PI3K-inhibitor LY 294002 (40 µM) for 45 minutes (D). (E) HEK293 cells were treated and Gab1 localisation analysed as described above in A but cultivated in 10% FCS or transfected with an expression vector for constitutive active PI3K (p110-CAAX). (F) As for E, HEK293 cells were cultivated in the absence of FCS (–, 1st lane), in the presence of 10% FCS (2nd lane), or transfected with an expression vector for constitutive active PI3K (p110g-CAAX) (3rd lane). To monitor PI3K activity, cell lysates were prepared and equal amounts of protein separated by SDS-PAGE. After western blotting, the membranes were stained for Akt phosphorylated at Ser473 to monitor PI3K activity. After stripping, the membrane was stained for Akt to ensure equal loading of the gel.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Constitutive membrane localisation of the isolated PH domain. (A) HEK293T cells were transfected with expression vectors for Gab1-GFP or the isolated PH domain fused to GFP together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day, cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes, the cells were stimulated with Epo (7 U/ml). Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation. (B) HEK293T cells were transfected with expression vectors for Gab1-GFP wild type or the mutants Gab1-PH-GFP, Gab1-PI3K-GFP, Gab1-Grb2-GFP or Gab1-SHP2-GFP, together with vectors encoding EpoR-gp130 chimeric receptors. Cells were stimulated and Gab1 localisation analysed as described in A.

View larger version (48K): [in this window] [in a new window]   Fig. 5. The region between L545 and E587 in Gab1 blocks membrane recruitment of full-length Gab1. (A) Schematic representation of the analysed Gab1-GFP fusion proteins used in this study. Tyrosine motifs involved in protein binding are indicated by Y. Proline-rich regions are highlighted in red. The Met-binding domain is underlined. The numbers represent the first and last amino acids in the mutant proteins corresponding to the numbering in Gab1 wild-type protein. GFP is fused to the C-terminus of Gab1. (B) HEK293T cells were transfected with expression vectors for Gab1-GFP fusion proteins depicted in A, together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes, the cells were stimulated with Epo (7 U/ml). Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Gab1 translocation to the plasma membrane is triggered by MAPK activity. (A) HEK293T cells were transfected with expression vectors for Gab1-GFP fusion proteins together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day, cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes, the cells were stimulated with Epo (7 U/ml) in the absence of the MEK inhibitor U0126 or after 15 minutes preincubation with 10 µM U0126. Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation. (B) To monitor efficient inhibition of ERK activation in U0126-treated cells, cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated STAT3 to control stimulation of the cells, and for the activated forms of ERK1/2. After stripping, the membrane was stained for STAT3 and ERK to monitor equal loading of the gel. (C) HEK293T cells were treated and Gab1 localisation analysed as described in A, except that cells were additionally transfected with vectors for the expression of constitutively active MEK or MEKK1. (D) To monitor efficient activation of ERK, cell lysates were prepared and proteins separated by SDS-PAGE. After western blotting, the membranes were stained for activated ERK (upper panel). After stripping, the membrane was stained for ERK to monitor equal loading of the gel. (E) HEK293T cells were treated and Gab1 localisation analysed as described above in A, except that cells were transfected with expression vectors for the wild-type chimeric receptor EpoR-gp130(YYYYYY), a corresponding receptor with a Y759F exchange EpoR-gp130(YFYYYY) or a receptor where all tyrosine residues within the cytoplasmic part of the receptor except Y759 were replaced by F.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Charged amino acids at residue 551 mimic the ERK stimulus to induce translocation of Gab1 to the plasma membrane. HEK293T cells were transfected with expression vectors for Gab1-GFP wild-type proteins or mutants where Ser551 and Ser567 are replaced by alanine or glutamate (A), or mutants where only Ser551 was replaced by alanine or glutamate (B), or mutants where only Ser567 was replaced by alanine or gluatmate (C) together with vectors encoding EpoR-gp130 chimeric receptors. 24 hours after transfection, cells were transferred to poly-L-lysine-coated coverslips. The next day, cells were starved for 3 hours in KRH and transferred into the perfusion chamber of a laser-scanning microscope. After 20 minutes the cells were stimulated with Epo (7 U/ml). Gab1 localisation is shown before stimulation and after 20 minutes of Epo stimulation.

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