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NOTCH1-IC suppresses PAK1 signaling pathway
through direct interaction
So-I Noh, Mi-Yeon Kim, Eun-Hye Jo, Hyung-Ju Lee, and Hee-Sae Park *
School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
Abstract Figure 1. NOTCH1-IC binds to PAK1 in intact Figure 4. NOTCH1-IC inhibits the interaction
cells.
between PAK1 and ILK1.
p21-Activated kinase 1 (PAK1) is a serine/threonine protein
kinase implicated in cytoskeletal remodeling and cell motility.[1]
Recent studies have shown that it also promotes cell proliferation,
regulates apoptosis, and increases cell transformation and
invasion. In this study, we shown that NOTCH1 intracellular
domain (NOTCH1-IC) negatively regulated PAK1 signaling
pathway. We found a novel interaction between NOTCH1-IC and
PAK1 both in vitro and in vivo. Overexpression of NOTCH1-IC
decreased PAK1 induced integrin linked kinase 1 (ILK1)
phosphorylation, whereas inhibition of NOTCH1 signaling
increased PAK1 induced ILK1 phosphorylation. Notably, ILK1
phosphorylation was higher in PS1/2-/- cells than in PS1/2+/+ (A) HEK293 cells were transiently transfected with the vector expressing GFP– (A) HEK293 cells were transfected with vectors expressing Myc–PAK1, Myc–
cells. As expected, overexpression of NOTCH1-IC decreased ILK1 PAK1 or Myc–NOTCH1-IC. After 48 h, the cells were lysed and the proteins in PAK1-CA, Myc–PAK1-KD, and Flag–NOTCH1-IC. After 48 h, the cells were lysed
induced phosphorylation of GSK-3 beta. Furthermore, NOTCH1- the cell lysates were immunoprecipitated using anti-Myc or anti-GFP antibody. and the proteins in the cell lysates were immunoprecipitated using anti-Myc
IC disrupted the interaction of PAK1 with ILK1 and altered PAK1 The immunoprecipitates were then immunoblotted using anti-GFP or anti-Myc antibody. The immunoprecipitates were then immunoblotted using anti-Flag
antibody. Expression of PAK1 or NOTCH1-IC was analyzed by immunoblotting
antibody. The cell lysates were also immunoblotted using anti-Flag and anti-
localization by directly interacting with it. This inhibitory effect of with anti-GFP or anti-Myc monoclonal antibody, respectively. (B) HEK293 cells GFP antibodies. (B) HEK293 cells were transiently transfected with an empty
NOTCH1-IC on the PAK1 signaling pathway was mediated by the were lysed, and the proteins in the cell lysates were immunoprecipitated using vector or vectors expressing HA–ILK1, GFP–PAK1, and Myc–NOTCH1-IC. After
binding of NOTCH1-IC to PAK1 and by the alteration of PAK1 anti-NOTCH1-IC antibody. The immunoprecipitates were then immunoblotted 48 h, the cells were lysed and the proteins in the cell lysates were
localization. Together, these results suggest that NOTCH1-IC is a with anti-PAK1 antibody. Expression of PAK1 or NOTCH1-IC was analyzed by immunoprecipitated using anti-HA antibody. The immunoprecipitates were
new regulator of the PAK1 signaling pathway that directly immunoblotting with anti-PAK1 or anti-NOTCH1-IC antibody, respectively. Cell then immunoblotted using anti-GFP antibody. The cell lysates were also
lysates were immunoblotted with anti-PAK1 and anti-NOTCH1-IC antibodies as
immunoblotted using anti-Myc, anti-HA, and anti-GFP antibodies. (C) HEK293
interacts with PAK1 and regulates its shuttling between the a control. (C) HEK293 cells were transfected with the vector expressing GFP– cells were transiently transfected with an empty vector or vectors expressing
nucleus and the cytoplasm. PAK1 or an empty vector. After 48 h, the cells were lysed and the cell lysates V5–ILK1, GFP–PAK1, and Myc–NOTCH1-IC. After 48 h, the cells were lysed and
were used for GST pull-down experiments by using GST or GST–NOTCH1-IC the proteins in the cell lysates were immunoprecipitated using anti-Myc
immobilized on GSH–agarose beads. Proteins bound to GST or GST–NOTCH1- antibody. The immunoprecipitates were then immunoblotted using anti-GFP
IC were analyzed by immunoblotting with anti-GFP antibody. The input and anti-V5 antibodies. The cell lysates were also immunoblotted using anti-V5,
represents 1% of the cell lysate before the in vitro binding assay. anti-GFP, and anti-Myc antibodies.
Introduction
p21-Activated kinase 1 (PAK1) is a serine/threonine (Ser/Thr) Figure 2. NOTCH1-IC decreases ILK1 Figure 5. NOTCH1-IC alters the localization of
protein kinase that is activated by external stimuli through
various cell surface receptors, including G-protein-coupled phosphorylation by PAK1. PAK1 and inhibits PAK-mediated cell migration.
receptors and receptor tyrosine kinases in a small GTPase-
dependent or GTPase-independent manner. PAK1 is a binding
partner of Rho GTPases Cdc42 and Rac1 and is involved in
diverse cellular processes such as cytoskeletal remodeling and
cell motility. Recent studies have shown that PAK1 also promotes
cell proliferation, regulates apoptosis, and increases cell
transformation and invasion. PAK1 is significantly overexpressed
in some cancers such as ovarian, breast, and bladder cancers.
Several functional studies have reported that PAK1 is highly
associated with cell transformation and tumorigenesis, as
evidenced by the development of premalignant lesions and tumor
formation due to PAK1 overexpression and hyperactivation. The
kinase activity of PAK1 is required for Ras-induced
transformation. Further, the activity of PAK1 is dependent on the
phosphorylation of Ser/Thr residues. To date, most studies have
focused on the cytosolic functions of PAK1. However, one study
showed that PAK1 binds to and phosphorylates histone H3 and
that endogenous PAK1 is localized in the nucleus of 18%–24%
interphase cells. Singh et al. identified the signaling sequences of (A) HEK293 cells were transiently transfected with the vector expressing GFP–
PAK1 or Myc–NOTCH1-IC. After 48 h, the level of ILK1 phosphorylation by
PAK1 that are involved in its nuclear localization and showed that PAK1 was measured by performing immunocomplex kinase assay. (B) HEK293
PAK1 regulates the expression of its targets in a positive as well cells were transiently transfected with the vector expressing Myc–NOTCH1-IC. (A) HEK293 cells were transfected with the vector expressing V5–ILK1, Myc–
PAK1-CA, or Myc–PAK1-KD. After 48 h, ILK1 and PAK1 were stained using
as negative manner. These findings highlight the opportunity to After 48 h, the cells were treated with EGF for 20 min at 37°C, and the level of Alexa 488 (green) and Alexa 532 (red) antibodies, respectively. (B) HEK293
determine new functions of PAK1 in the nucleus, including its ILK1 phosphorylation by PAK1 was measured by performing immunocomplex cells were transfected with the vector expressing V5–ILK1, GFP–PAK1, or
possible role in cell cycle regulation, mitosis, and cancer. PAK1 is kinase assay. (C) HEK293 cells were transiently transfected with the vector Myc–NOTCH1-IC. After 48 h, ILK1 was stained using Alexa 532 (red) antibody.
expressing GFP–PAK1. After 48 h, the cells were treated with DAPT for 6 h at
involved in the regulation of cell cycle, and its overexpression in 37°C, and the level of ILK1 phosphorylation by PAK1 was measured by (C) HEK293 cells were transfected with vectors expressing GFP–PAK1 and
human breast cancer cells results in the abnormal accumulation performing immunocomplex kinase assay. (D) HEK293 cells were first Myc–NOTCH1-IC. After 48 h, PAK1 and NOTCH1-IC were stained using Alexa
488 (green) and Alexa 532 (red) antibodies, respectively. (D) HeLa cells were
of centrosomes and aberrant mitosis. Recent studies have shown transfected with NOTCH1 shRNA (pSUPER-shNOTCH1) or control shRNA transfected with the expressing NOTCH1-IC and PAK1. After 48 h of
that functions of PAK1 may be regulated by its intracellular (pSUPER-shCon) and then with the vector expressing GFP–PAK1. After 48 h, transfection, cell monolayers were wounded using a sterile 200-μl pipette tip
location. While the role of PAK1 in the cytoplasm is well the level of ILK1 phosphorylation by PAK1 was measured by performing and were washed with the culture medium. The cells were photographed at 0 h
immunocomplex kinase assay. (A–D) Cell lysates were also immunoblotted
established, its role in the nucleus is unknown. Studies indicate using the indicated antibodies. Immunocomplex PAK1 kinase assays were and were incubated in a complete medium for 12 , 24, and 36 h. Quantification
that PAK1 localizes to and performs several functions within the conducted using His–ILK1 as the substrate. was performed by measuring the distance migrated by the transfected cells
compared with that by control cells.
nucleus. However, the mechanisms regulating the localization of
PAK1 to the nucleus are unknown.
Integrin-linked kinase 1 (ILK1) decreases the stability of NOTCH1
intracellular domain (NOTCH1-IC) through Fbw7 ubiquitin ligase-
mediated degradation via ubiquitin–proteasome pathway.[2] ILK1 Figure 3. NOTCH1-IC inhibits ILK-mediated Conclusion
is a pivotal effector in various cellular processes such as cell phosphorylation of GSK-3 beta.
migration, invasion, proliferation, differentiation, metabolism, and
survival.[3] Studies have shown that ILK1 regulates cell motility In this study, we observed a novel interaction between
and migration through the small GTPase Rac1 and Cdc42. PAK1 NOTCH1-IC and PAK1 both in vitro and in vivo. We also
and ILK1 regulate some common physiological processes. ILK1 observed that PAK1 decreased ILK1 phosphorylation by
is a substrate of PAK1 in cellular processes such as cytoskeletal NOTCH1-IC and increased the nuclear localization of PAK1
remodeling, cell motility, proliferation, apoptosis, transformation, through interaction with NOTCH1-IC. We identified that
and invasion. Studies have reported that ILK1 undergoes NOTCH1-IC was a physiological binding partner of PAK1
phosphorylation-dependent shuttling between the nucleus and and inhibited the oncogenic PAK1 signaling pathway.
the cytoplasm. Chun et al. suggested that ILK1 localizes to the
nucleus through a putative nuclear localization signal.[4]
Previously, we reported the colocalization of ILK1 and NOTCH1-
IC in both the nucleus and the cytoplasm of HaCaT and
melanoma cells. ILK1 controls tumor growth and angiogenesis by References
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NOTCH1-IC and PAK1 signaling and observed a novel interaction (A) HEK293 cells were transiently transfected with the vector expressing GFP– Biol Chem 2000, 275:12041-12050.
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the kinase activity of ILK1. Notably, NOTCH1-IC disrupted the After 48 h, the cells were treated with EGF for 20 min at 37°C, and the level of Notch1 signaling by down-regulation of protein stability
interaction of PAK1 with ILK1 and altered the localization of PAK1. ILK1 phosphorylation by PAK1 was measured by performing immunocomplex through Fbw7 ubiquitin ligase. Mol Cell Biol 2007, 27:5565-5574.
This inhibitory effect of NOTCH1-IC on the ILK1 signaling kinase assay. (C) HEK293 cells were transiently transfected with the vector [3] Hannigan G, Troussard AA, Dedhar S: Integrin-linked kinase:
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that directly interacts with PAK1 and regulates its shuttling (pSUPER-shCon) and then with the vector expressing GFP–PAK1. After 48 h, Phosphorylation-dependent regulation of nuclear localization
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PAK1-mediated signaling. conducted using His–ILK1 as the substrate.
