Supplementary Figure 1, The GST fusion proteins were analyzed by immunoblotting with antiGST antibody.
Supplementary Figure 2, a, A point mutation (P842L) in PLC- diminishes its association with
PIKE. Five g each purified GST-PLC--SH3 and GST-PLC--SH3 (P842L) incubated with
HEK 293 cell lysate, transfected with Myc-PIKE, at 4 oC for 2 hr. The samples were
precipitated with Glutathione Sepharose 4B beads and analyzed by Western blot using anti-Myc
antibody. Similar levels of GST-PLC--SH3 and GST-PLC--SH3 (P842L) proteins were
employed in the experiments. b, The SH3 domain is required for PLC- 's GEF activity. The
constructs used are GST-SH223 (a GST construct containing the two SH2 and the SH3 domain
of PLC-), GST-SH22 (a GST construct containing the two SH2 domains of PLC-) and GSTSH3 (a GST construct containing the SH3 domain of PLC-), (Upper left). GST-SH223 and
GST-SH3 but not GST-SH22 bind strongly to PIKE (Upper right). Similar levels of various
GST fusion proteins were employed (lower left). SH223 and SH3 GST fusion proteins strongly
stimulate the dissociation of [3H] GDP (Lower right), consistent with their strong interaction
with PIKE (Upper right). In contrast, GST-SH22 only weakly influences dissociation [3H] GDP
from PIKE, consistent with its diminished binding activity to PIKE. The GDP release
experiment, conducted the same as for figure 2b, employed purified GST-PLC--SH223 (and
GST-PLC--SH3 () GST-PLC--SH22 (). c, GST-PLC--SH223 binds predominantly to
PIKE in the presence of GDP, while GTP inhibits their interaction. GST-PLC--SH223 and
HEK 293 cell lysate, which were transfected with Myc-PIKE, were incubated at 4°C for 2 hr.
The incubation was performed in the presence or absence of guanine nucleotide as indicated.
Similar levels of all GST-PLC--SH223 proteins were employed in all experiments.
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Supplementary Figure 3, Upper Left: GST-PIKE-PRD (350-370) and GST-PIKE (262-384)
fusion proteins were coupled to glutathione-Sepharose beads and incubated with HEK 293 cell
lysates, transfected with PLC-1 or PLC--SH223 plasmids. The beads were washed
extensively and bound proteins eluted by boiling in Laemmli sample buffer. The samples were
resolved by SDS-PAGE on a 4-12% gel, and visualized by immunoblotting with anti-PLC-1
antibody. The two PIKE constructs bind to wild-type PLC- but not to PLC--SH223. Lower
left: In cells transfected with PLC--SH223 (right lane), expression is detected both for
endogenous PLC- and PLC--SH223, while in cells transfected with wild-type PLC- (left
lane), we detect expression of the transfected wild-type PLC-. Right: GST-PLC--SH3 directly
associates with His-PIKE (262-384) but not with a His-NuMA fragment employed as control.
GST-PLC--SH3 beads were incubated with His-PIKE (262-384) or an unrelated control protein
His-NuMA (1440-1913), and the washed beads were electrophoresed and stained. PLC--SH3
binds to His-PIKE (262-384) but not His-NuMA (1440-1913).
Supplementary Figure 4 a, Endogenous PLC-1 localizes to both cytoplasm and nucleus of
PC12 cells. b, PLC- in stably transfected PC12 cells penetrates to the nucleus, though
predominantly locates in the cytoplasm, and more translocate to the nucleus in response to NGF
treatment as the native cells.
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