To understand the molecular mechanism by which the IQ peptide inhibited cancer
metastasis, SW620 cells were treated by the PepIQ or a control peptide. Cell proliferation and
cell migration were analyzed. Clearly, the PepIQ did not affect cell proliferation, while cell
migration was almost completely abolished under the peptide treatment (Fig. 4.1D and Fig. 4.1E).
Similarly, cell migration was also dramatically inhibited by stableexpression of the eEGF-IQ in
SW480 cells (Fig. S34.2A). We questioned why the peptide PepIQ was so effective in inhibiting
cell migration. It was demonstrated that p68 interacts with the calmodulin in a number of in vitro
binding studies and the IQ motif covered by the PepIQ harbors the p68- calmodulin interaction
site (Shen, Valencia et al. 2005; Jang, Guo et al. 2007). Thus, it is possible that the PepIQ
competed with p68 to interact with calmodulin in the cells therefore inhibited cell migration.
Thus, we sought to test whether the IQ peptide interrupts the p68-calmodulin interaction and
consequently inhibits cell migration. We first probed the p68 and calmodulin interaction by
carrying out co-precipitation experiments with cell extracts made from colon cancer cells SW480
using calmodulin beads. Clearly, p68 was precipitated down from the cellular extracts by the
motif of p68 in the calmodulin binding, we created a mutant with mutation (IQ – MA, ref to as
IQ-M). The calmodulin bead pull-down demonstrated that the mutant interacted with calmodulin
with a substantially weak affinity, and the IQ-M: calmodulin interaction was Ca2+ independent
(Fig. 4.2B). The results indicated that the strong Ca2+-dependent p68-calmodulin interaction is
IQ motif-dependent.
Since we observed that the PepIQ greatly inhibited cell migration, we reasoned whether
there is a cell migration signal induced p68-calmodulin interaction and the peptide may interrupt
the migration induced p68-calmodulin interaction. Thus, we first examined the p68 and
calmodulin interaction under various cell migration stimuli. It is well known that treatment of
cells with EGF can induce cell migration. Thus, we first carried out co-immunoprecipitation with
cellular extracts made from SW480 cells using an antibody against calmodulin. The cells were
pre-treated with EGF. There was a strong increase in the p68 and calmodulin co-precipitation
upon the EGF treatment (Fig. S4.3A and Fig. S4.3B). Measurement of the changes in cell
migrations under the stimulation of EGF revealed a close correlation between the increase in the
p68 and calmodulin interaction and cell migration (Fig. S4.1C). We next probed the p68 and
calmodulin interaction by the co-immunoprecipitation with cell extracts prepared from SW480
cells that were subjected to multiple scratch-wound treatment. A strong increase in the p68 and
calmodulin co-immunoprecipitation was observed and the increase depended upon the number of
scratches (Fig. 4.2E). The results suggest cell migration induced p68 and calmodulin interaction.
Since the IQ motif is the site for the strong Ca2+-dependent p68 and calmodulin interaction, we
asked whether the increase in the p68 and calmodulin interaction induced by cell migration
requires the IQ motif. HA-IQ-M was exogenously expressed in SW480 cells in which the
enhancement was not observed in the IQ-M mutant expressed cells (Fig. S4.3D). To test whether
the peptide abolished the inducible p68-calmodulin interaction upon cell migration, we probed
the p68-calmodulin interaction in cell lysate prepared from SW480 cells that were transiently
expressed with eGFP-IQ. The p68-calmodulin interaction was not strengthened upon migration
stimuli in SW480 cells in which the eGFP-IQ was stably expressed (Fig. S4.2B). Clearly, our
experiments supported the notion that the cell migration induced p68 and calmodulin interaction,
and the IQ peptide interrupted the induced p68-calmodulin interaction thus inhibited cell
migration.
Based on the preceding experiments, we suspected that the p68-calmodulin interaction
would be critical for cell migration. The hypothesis was tested by both boyden chamber assay
and scratch-wound assay. The endogenous p68 was knocked down in SW480 cells and HA-p68s,
wt, IQ-M, LGLD (An ATPase activity deficiency mutant (Lin, Yang et al. 2005)), or Y593F
(Yang, Lin et al. 2006), was expressed in the cells (Fig. S4.3E). Boyden chamber assays showed
that knockdown of p68 resulted in dramatic reduction in cell migration and the cell migration
could be fully recovered by reexpression of wt p68, but not by the IQ-M mutant. As a control,
the cell migration could also be recovered by expression of a mutant that carries mutation at an
irrelevant site (Y595F) (Fig. 4.3A). Interestingly, expression of an ATPase deficiency mutant
LGLD in the p68 knockdown cells did not recover cell migration, indicating that the ATPase
activity of p68 is required for cell migration (Fig. 4.3A). We further confirmed the functional
role of the p68 and calmodulin interaction in cell migration by the scratch-wound assay. Scratch-
wound was introduced to the plates that cultured SW480 cells expressing p68 wt, IQ-M, LGLD,
or Y593F. The amount of cells expressing IQ-M or LGLD migration to the wound was much
HA-tag revealed that no IQ-M and LGLD expressing cells migrated to the wounds, while p68 wt
expressing cells migrated to the wound areas normally (Fig. 4.3C). These experiments showed
that the p68-calmodulin interaction is required for cell migration.