Bifurcation of cell migratory and proliferative signaling by the adaptor protein Shc - PubMed (original) (raw)

Bifurcation of cell migratory and proliferative signaling by the adaptor protein Shc

L R Collins et al. J Cell Biol. 1999.

Abstract

Cytokines and extracellular matrix proteins initiate signaling cascades that regulate cell migration and proliferation. Evidence is provided that the adaptor protein Shc can differentially regulate these processes. Specifically, under growth factor-limiting conditions, Shc stimulates haptotactic cell migration without affecting anchorage-dependent proliferation. However, when growth factors are present, Shc no longer influences cell migration; rather, Shc is crucial for DNA synthesis. Mutational analysis of Shc demonstrates that, while tyrosine phosphorylation is required for both DNA synthesis and cell migration, the switch in Shc signaling is associated with differential use of Shc's phosphotyrosine interacting domains; the PTB domain regulates haptotaxis, while the SH2 domain is selectively required for proliferation.

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Figures

Figure 1

Figure 1

Decreased actin organization and increased migration of Shc expressing Cos-7 cells. (Left) Serum-deprived control and Shc stably expressing cells were harvested, plated on collagen-coated coverslips for 2 h, fixed, permeabilized, and stained with rhodamine phalloidin. The images shown are single confocal optical sections taken with a 63× objective (left). The scale bar is equal to 50 μm. (Right) Migration of parental vs Shc-expressing Cos-7 cells. Serum-deprived cells were allowed to migrate for 4 h in Boyden chambers towards a collagen matrix. Data from one of four independent experiments are shown (mean ± SE). The enhanced migration of the Shc cell line was found to be statistically significant (P < 0.05 by Student's t test). Random migration towards BSA-coated chambers was always <2% and subtracted from each value.

Figure 1

Figure 1

Decreased actin organization and increased migration of Shc expressing Cos-7 cells. (Left) Serum-deprived control and Shc stably expressing cells were harvested, plated on collagen-coated coverslips for 2 h, fixed, permeabilized, and stained with rhodamine phalloidin. The images shown are single confocal optical sections taken with a 63× objective (left). The scale bar is equal to 50 μm. (Right) Migration of parental vs Shc-expressing Cos-7 cells. Serum-deprived cells were allowed to migrate for 4 h in Boyden chambers towards a collagen matrix. Data from one of four independent experiments are shown (mean ± SE). The enhanced migration of the Shc cell line was found to be statistically significant (P < 0.05 by Student's t test). Random migration towards BSA-coated chambers was always <2% and subtracted from each value.

Figure 2

Figure 2

Shc stimulates migration on collagen and vitronectin. (Top) Cos-7 cells were either mock transfected (pcDNA3.1HisC plus lac z) or transfected with p52 Shc cDNA plus lac z. Cells were deprived of serum and migration was assessed as indicated with either migration media (mock, p52) or 100 ng/ml EGF (Mock+EGF) in the lower chamber. Migration was quantitated by enumerating the number of β-galactosidase positive cells/field using a 20× objective. The mean ± SE from a representative of five independent experiments is shown. (Bottom) Tyrosine phosphorylation of p52 Shc in response to integrin ligation. Serum-deprived cells expressing p52 Shc were harvested, then either held in suspension or replated on collagen or vitronectin for 1 h and lysed in RIPA buffer. His-tagged Shc was isolated with nickel agarose beads and analyzed for phosphotyrosine content by Western blot (mAb 4G10, upper autoradiogram). The blot was then stripped and reprobed with an anti-Shc antibody as described in Materials and Methods (lower autoradiogram).

Figure 3

Figure 3

Shc stimulates haptotactic migration. (Top) Shc-stimulated cell migration is integrin dependent. Cells were processed for migration assay as described in Materials and Methods, except that cells were mixed with anti-integrin monoclonal antibodies as indicated before loading into Boyden chambers. Next, cells were allowed to migrate for 4 h and quantitated for migration as described above. Each bar represents the mean ± SE from one experiment representative of three with similar results. (Bottom) Serum-deprived mock or p52 Shc transfected Cos-7 cells were allowed to migrate on Boyden chambers coated on either their lower surface (haptotaxis) or both their upper and lower surfaces (random migration). Each bar represents the mean ± SE of a representative experiment performed in triplicate.

Figure 4

Figure 4

Schematic of Shc constructs. Full-length p52 Shc is depicted. Gray, white, and hatched regions represent the NH2-terminal PTB domain, the CH domain, and the SH2 domain respectively. The point mutations used are indicated above the schematic. Mutants are identified by S154P, Y239/Y240F, Y317F, Y239/Y240/Y317F (Shc 3YF), and R401L.

Figure 5

Figure 5

Mutational analysis of Shc-stimulated migration. (A) Cos-7 cells were transfected with increasing amounts of the indicated Shc cDNAs (0.1, 0.3, and 3.0 μg/10-cm plate), serum deprived and assessed for haptotaxis towards collagen. Each bar represents the mean ± SE of a representative experiment. Lysates were generated from the cells used in these experiments and analyzed by immunoblotting with polyclonal anti-Shc antibodies. His-tagged Shc can be discerned from endogenous Shc in the Western blot by its slightly retarded mobility on the gel. The 0.3 μg/plate condition was repeated at least five times for all the mutants with similar results. Cell migration induced by both p52wt and R401L was found to be highly statistically significant (P < 0.01 by ANOVA). (B) tyrosine phosphorylation pattern of Shc mutants. Cells were either held in suspension or plated on collagen or vitronectin. As a positive control, cells were stimulated with insulin as indicated. His-tagged Shc was isolated after lysis in RIPA buffer by incubation with nickel agarose beads and analyzed by Western blotting with anti-phosphotyrosine. Blots were then stripped and reprobed with pAb Shc.

Figure 5

Figure 5

Mutational analysis of Shc-stimulated migration. (A) Cos-7 cells were transfected with increasing amounts of the indicated Shc cDNAs (0.1, 0.3, and 3.0 μg/10-cm plate), serum deprived and assessed for haptotaxis towards collagen. Each bar represents the mean ± SE of a representative experiment. Lysates were generated from the cells used in these experiments and analyzed by immunoblotting with polyclonal anti-Shc antibodies. His-tagged Shc can be discerned from endogenous Shc in the Western blot by its slightly retarded mobility on the gel. The 0.3 μg/plate condition was repeated at least five times for all the mutants with similar results. Cell migration induced by both p52wt and R401L was found to be highly statistically significant (P < 0.01 by ANOVA). (B) tyrosine phosphorylation pattern of Shc mutants. Cells were either held in suspension or plated on collagen or vitronectin. As a positive control, cells were stimulated with insulin as indicated. His-tagged Shc was isolated after lysis in RIPA buffer by incubation with nickel agarose beads and analyzed by Western blotting with anti-phosphotyrosine. Blots were then stripped and reprobed with pAb Shc.

Figure 6

Figure 6

Shc requirement for haptotaxis in a metastatic cell line. (A) Shc is constitutively phosphorylated in human FG-M pancreatic carcinoma cells. Serum-deprived FG-M cells were either held in suspension or plated on extracellular matrix proteins as indicated for 30 min. Cells were then lysed and analyzed by immunoblotting with anti-phosphotyrosine as described in Materials and Methods. (B) FG-M cells require Shc tyrosine phosphorylation and the Shc PTB domain for haptotaxis. FG-M cells were transfected as indicated and assayed for haptotaxis on vitronectin by counting the number of transfected cells/high powered field. Each bar represents the mean ± SE from a single representative experiment performed in triplicate.

Figure 7

Figure 7

Shc is not required for EGF-stimulated cell migration. Cos-7 cells were transfected with cDNAs encoding Shc mutants as indicated, serum deprived, and assayed for migration in the presence of 100 ng/ml EGF as described in Materials and Methods. The data are depicted as relative migration, with the migration induced by the indicated forms of Shc in the absence of EGF defined as one. Each bar represents the mean ± SE of four independent experiments performed in duplicate. None of the differences between the groups were found to be statistically significant.

Figure 8

Figure 8

Shc is required for EGF to stimulate anchorage-dependent DNA synthesis. Cos-7 cells were transfected with the indicated cDNAs, serum deprived and assayed for BrdU incorporation in either the absence (top) or the presence of 100 ng/ml EGF (bottom). The data are presented as the percentage of transfected cells incorporating BrdU (top) or as percent increase in BrdU incorporation stimulated by the addition of EGF (bottom). Each bar represents the mean ± SD of three independent experiments performed in triplicate.

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