Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 8:18:1-12.
doi: 10.2142/biophysico.bppb-v18.001. eCollection 2021.

Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation

Ryo Yoshizawa  1   2 , Nobuhisa Umeki  1 , Akihiro Yamamoto  1 , Masayuki Murata  2 , Yasushi Sako  1
Affiliations

Biphasic spatiotemporal regulation of GRB2 dynamics by p52SHC for transient RAS activation

Ryo Yoshizawa et al. Biophys Physicobiol. .

Abstract

RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.

Keywords: FCCS; RTK-RAS-MAPK; cell signaling; single-molecule imaging; temporal regulation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Protein expression and phosphorylation dynamics. (A) The expression profiles of GFP-GRB2 (upper panel), Halo-SHC or Halo-SHC3F (middle panel), and the corresponding endogenous proteins under the indicated cell conditions were assessed by western blotting analysis of cell lysates with the indicated antibodies (Ab). MW, molecular weight markers. (B) Changes in GRB2 and SHC expression after HRG stimulation (0, 5, and 30 min). Values were normalized as described in the Methods section. The expression levels of GFP-GRB2 and Halo-SHC were normalized using the corresponding endogenous protein levels without stimulation, respectively. (C) Schematic structures of the SHC constructs. The HaloTag was fused to the N-termini of the constructs. Numbers represent the amino acid positions. (D, E) Time course analysis of the ERBB receptor (D) and SHC (E) phosphorylations after HRG stimulation (0, 5, and 30 min). Phosphorylations shown in the upper and lower panels in (D) are those of the major SHC and GRB2 binding sites, respectively. Values were normalized as described in the Methods section. In (B, D, E), the mean values for three independent experiments were plotted along with the standard error. Asterisks denote statistical significance against the values obtained in control cells expressing WT-SHC (p<0.05 by t test).
Figure 2
Figure 2
Membrane translocation of GRB2 and SHC. (A) Epi-fluorescence (Epi) and TIRF (TIR) images of GFP-GRB2 and Halo-SHC in MCF7 cells. Scale bar,10 μm. (B) Time course of GFP-GRB2 (left) and Halo-SHC (right) translocations to the plasma membrane. Cells were stimulated with 10 nM HRG at time 0. Cells co-expressing GFP-GRB2 and WT (green) or 3F mutant (orange) of Halo-SHC and expressing GFP-GRB2 with an SHC-knockdown (blue) were examined. Vertical axes were normalized to the values obtained before stimulation. The mean values for 16–32 cells were plotted along with the standard errors. (C) Frequencies of appearance of GFP-GRB2 molecules (relative numbers/sec/100 μm2) on the plasma membrane were plotted against the relative expression level of GFP-GRB2 observed by epi-illumination. Results of linear fitting (lines) are shown with the correlation coefficient, r. (D) Relative association rate constants for GRB2. (E) Dwelling time distributions of single GFP-GRB2 particles on the basal plasma membrane. (F) Dissociation rate constants for the fast (k1, left) and slow (k2, right) dissociation components of GRB2. (G) Fraction sizes for the slow dissociation component of GRB2. Cells in (C-G) were co-expressed with GFP-GRB2 and either WT SHC or its 3F mutant. In (D-G), the mean values before (black), and at 5 min (red), and 30 min (blue) after HRG stimulation are shown with standard errors. Number of cells examined were 21–24 (D), and 27–32 (E-G). Asterisks denote statistical significance (p<0.05 by t test).
Figure 3
Figure 3
Cytoplasm interaction of GRB2 and SHC. (A) Schematic representation of FCCS measurement (left) and fluorescence micrographs of a cell co-expressing GFP-GRB2 and Halo-SHC (middle). Fluctuations in the fluorescence intensities (count rate) in a detection area (arrows in the middle) were detected for FCCS analysis (right). Scale bar, 10 μm. (B) Typical autocorrelation (left) for GFP-GRB2 (green) and Halo-SHC (red), or cross-correlation (right) curves in the same detection volumes. Curves were fitted (blue lines) with functions described in the Methods section. (C) The relative concentrations of GFP-GRB2 (upper) and Halo-SHC (WT or 3F, lower) were determined from the autocorrelation curves. (D, E) The dissociation constant (Kd) of GRB2/SHC complex (D) and fraction ratios of the complex to the total GRB2 or SHC levels (E) were calculated from the FCCS analysis. The mean values for 14–16 cells are plotted along with the standard errors. Asterisks denote statistical significance (p<0.05 using the t test).
Figure 4
Figure 4
SOS and RAF translocation and effects of a MEK inhibitor. (A) Membrane translocation time courses of GFP-SOS (left) and GFP-RAF (right) in cells co-expressing Halo-SHC (green) or Halo-SHC3F (orange), or with an SHC-knockdown (blue). (B, C) Membrane translocation time courses of GFP-GRB2, Halo-SHC, GFP-SOS, and GFP-RAF in cells pretreated with 10 μM U0126. GRB2, SOS, and RAF were co-expressed with Halo-SHC (green) or Halo-SHC3F (orange). (D) Membrane translocation time courses of SOS R1131K-Halo in cells with co-expression of WT-SHC (green) and SHC3F (orange). Vertical axes were normalized to the values obtained before stimulation. The mean values for 8–40 (A) or 13–30 (B, C) or 8–16 (D) cells were plotted along with the standard errors.
Figure 5
Figure 5
Biphasic spatiotemporal regulation of GRB2 by SHC. Recognizing the phosphorylation sites (P) on the activated EGFR molecules, GRB2 and SHC are translocated to the plasma membrane. At the early stage (<10 min), SHC increases the density of GRB2/SOS complex on the plasma membrane to increase the amplitude of RAS activity. Whereas, at the later stage (30 min), SHC inhibits the membrane translocation of GRB2 through the complex formation in the cytoplasm and suppresses RAS activation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Marshall, C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179–185 (1995). DOI: 10.1016/0092-8674(95)90401-8 - PubMed
    1. Kao, S., Jaiswal, R. K., Kolch, W. & Landreth, G. E.. Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells. J. Biol. Chem. 276, 18169–18177 (2001). DOI: 10.1074/jbc.M008870200 - PubMed
    1. Nagashima, T., Shimodaira, H., Ide, K., Nakakuki, T., Tani, Y., Takahashi, K., et al. . Quantitative transcriptional control of ErbB receptor signaling undergoes graded to biphasic response for cell differentiation. J. Biol. Chem. 282, 4045–4056 (2007). DOI: 10.1074/jbc.M608653200 - PubMed
    1. Sharma, S. V., Bell, D. W., Settleman, J. & Haber, D. A.. Epidermal growth factor receptor mutations in lung cancer. Nat. Rev. Cancer 7, 169–181 (2007). DOI: 10.1038/nrc2088 - PubMed
    1. Lake, D., Correa, S. A. & Muller, J.. Negative feedback regulation of the ERK1/2 MAPK pathway. Cell. Mol. Life Sci. 73, 4397–4413 (2016). DOI: 10.1007/s00018-016-2297-8 - PMC - PubMed