Requirement for phospholipase C-gamma1 enzymatic activity in growth factor-induced mitogenesis

Abstract

The cytoplasmic regions of the receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) bind and activate phospholipase C-gamma1 (PLC-gamma1) and other signaling proteins in response to ligand binding outside the cell. Receptor binding by PLC-gamma1 is a function of its SH2 domains and is required for growth factor-induced cell cycle progression into the S phase. Microinjection into MDCK epithelial cells and NIH 3T3 fibroblasts of a polypeptide corresponding to the noncatalytic SH2-SH2-SH3 domains of PLC-gamma1 (PLC-gamma1 SH2-SH2-SH3) blocked growth factor-induced S-phase entry. Treatment of cells with diacylglycerol (DAG) or DAG and microinjected inositol-1,4,5-triphosphate (IP3), the products of activated PLC-gamma1, did not stimulate cellular DNA synthesis by themselves but did suppress the inhibitory effects of the PLC-gamma1 SH2-SH2-SH3 polypeptide but not the cell cycle block imposed by inhibition of the adapter protein Grb2 or p21 Ras. Two c-fos serum response element (SRE)-chloramphenicol acetyltransferase (CAT) reporter plasmids, a wild-type version, wtSRE-CAT, and a mutant, pm18, were used to investigate the function of PLC-gamma1 in EGF- and PDGF-induced mitogenesis. wtSRE-CAT responds to both protein kinase C (PKC)-dependent and -independent signals, while the mutant, pm18, responds only to PKC-independent signals. Microinjection of the dominant-negative PLC-gamma1 SH2-SH2-SH3 polypeptide greatly reduced the responses of wtSRE-CAT to EGF stimulation in MDCK cells and to PDGF stimulation in NIH 3T3 cells but had no effect on the responses of mutant pm18. These results indicate that in addition to Grb2-mediated activation of Ras, PLC-gamma1-mediated DAG production is required for EGF- and PDGF-induced S-phase entry and gene expression, possibly through activation of PKC.

FIG. 1

GST fusion PLC-γ1 proteins used in this study. SH2 domains (shaded bars), SH3 domains (empty bars), and catalytic phospholipase domains (solid bars) are indicated.

FIG. 2

Association of GST fusion PLC-γ1 proteins with the activated EGFRs, Sos, and dynamin in vitro. GST fusion PLC-γ1 proteins were bound to glutathione-Sepharose beads and incubated with lysates from EGF-stimulated MDCK cells. Bound proteins were detected by Western immunoblotting with anti-EGFR, anti-Sos, or antidynamin antibodies. Lanes: 1, PLC-γ1 SH2-SH2-SH3; 2, PLC-γ1 SH2-SH2; 3, PLC-γ1 NSH2; 4, PLC-γ1 CSH2; 5, PLC-γ1 SH3; 6, GST.

FIG. 3

Inhibition of EGF-induced DNA synthesis by GST fusion PLC-γ1 SH3 and SH2 domains. Quiescent MDCK cells were microinjected with the indicated GST fusion proteins, and EGF (100 ng/ml) and BrdU were added to the incubation medium. The cells were fixed and stained after 16 h of treatment. The percentage of BrdU-positive cells was calculated as described in Materials and Methods. Data are means + standard errors of three independent experiments.

FIG. 4

The inhibition of S-phase entry by microinjection of PLC-γ1 SH2-SH2-SH3 but not Grb2 SH2 or anti-Ras antibody is relieved by IP₃ and DAG. Quiescent MDCK cells were injected with the proteins indicated below, incubated at 37°C for 1 h, and then incubated with EGF (100 ng/ml) and BrdU with or without DAG for 15 h. Following fixation, the microinjected cells were identified by use of either FITC-conjugated avidin (A to D) or FITC-conjugated antirat antibody (E and F), and BrdU incorporation was detected by use of mouse anti-BrdU antibody followed by TRITC-conjugated antimouse antibody. (A) Cells injected with PLC-γ1 SH2-SH2-SH3; (B) cells injected with PLC-γ1 SH2-SH2-SH3 and IP₃ and treated with DAG1; (C) cells injected with Grb2 SH2; (D) cells injected with Grb2 SH2 and IP₃ and treated with DAG1; (E) cells injected with anti-Ras Y13-259; (F) cells injected with anti-Ras Y13-259 and IP₃ and treated with DAG1. Magnification, ×120.

FIG. 5

EGF-induced S-phase entry is restored by IP₃ and DAG in cells injected with PLC-γ1 SH2-SH2-SH3 but not in cells injected with Grb2 SH2 or anti-Ras. Quiescent MDCK cells were injected with the proteins indicated at the bottom of the figure, incubated at 37°C for 1 h, incubated with EGF (100 ng/ml) and BrdU with or without DAG for 15 h, and then processed for immunofluorescence. The percent BrdU-positive cells was calculated as described in Materials and Methods. Data are means + standard errors of three independent experiments.

FIG. 6

Relief of the PLC-γ1 SH2-SH2-SH3 inhibition of EGF-induced S-phase entry by DAG. Quiescent MDCK cells were injected with PLC-γ1 SH2-SH2-SH3, incubated at 37°C for 1 h, and then incubated with EGF (100 ng/ml) and BrdU with or without DAG for 15 h. The cells were fixed and processed for immunofluorescence, and the percentage of BrdU-positive cells was calculated as described in Materials and Methods. Data are means + standard errors of three independent experiments.

FIG. 7

Microinjection of PLC-γ1 SH2-SH2-SH3 inhibits EGF-induced PKC-dependent, but not PKC-independent, activation of c-fos promoter plasmids. Quiescent MDCK cells were injected with wtSRE-CAT (A to F) or mutant pm18 (G to L) together with PLC-γ1 SH2-SH2-SH3 (C to F and I to L) or GST (A, B, G, and H) and incubated at 37°C for 1 h, incubated with EGF (100 ng/ml) with (E, F, K, and L) or without (A to D and G to J) DAG for 15 h, and then fixed and stained by immunofluorescence. Microinjected cells were identified with an FITC-avidin stain (left panels), and CAT was assayed with polyclonal anti-CAT antibody followed by a TRITC-conjugated antirabbit antibody (right panels). Magnification, ×120.

FIG. 8

Microinjection of PLC-γ1 SH2-SH2-SH3 inhibits EGF- and PDGF-induced PKC-dependent, but not PKC-independent, activation of c-fos promoter plasmids. Quiescent MDCK cells (A) or NIH 3T3 cells (B) were injected with wtSRE-CAT (unshaded) or mutant pm18 (shaded) plasmids together with GST or PLC-γ1 SH2-SH2-SH3 and incubated at 37°C for 1 h, incubated with EGF (100 ng/ml) or PDGF (20 ng/ml) with or without DAG for 15 h, and then fixed and stained by immunofluorescence. Microinjected cells were identified with an FITC-avidin stain, and CAT was assayed with a polyclonal anti-CAT antibody followed by a TRITC-conjugated antirabbit antibody. The percentage of cells positive for CAT expression was calculated as described in Materials and Methods. Data are means + standard errors of three independent experiments.