RASSF1A Is Part of a Complex Similar
to the Drosophila Hippo/Salvador/Lats
Cai Guo, Stella Tommasi, Limin Liu, Jiing-Kuan Yee,
Reinhard Dammann, and Gerd P. Pfeifer
Supplemental Experimental Procedures
Plasmids and Antibodies
pEBG and pEBG-RASSF1A have been described previously [S1].
derivatives (MST2-K56R), were kindly provided by J. Avruch
(Massachusetts General Hospital). pCGN-HA-LATS1 wild-type and
kinase-dead derivatives (K734A) were kindly provided by H. Saya
(Kumamoto University). Full-length WW45 (cDNA from ATCC) was
cloned into pcDNA3/Flag, and pcDNA3/HA vectors were obtained
from David Stern (Yale University). DNA fragments corresponding
to amino acids 199–383, 1–267, and 1–320 of WW45 were subcloned
into the pcDNA3/HA vector. Full-length RASSF1A as well as the
indicated deletion constructs were cloned into the pcDNA3/Flag
vector or the pEBG vector.
RASSF1A antibody (M304) was prepared with RASSF1A N-termi-
antigen injected into rabbits. WW45 antibody (#4) was prepared with
WW45 sequences (amino acids 268–383) fused to a GST tag for use
as antigen injected into rabbits. Other antibodies were from
Figure S1. Binding-Domain Mapping of RASSF1A on WW45
A diagram of the human WW45 protein and its domains is shown at the top. Left panels: different deletion constructs of HA-WW45 were cotrans-
fected with full-length GST-RASSF1A (5 mg each) into COS7 cells. Protein complexes were isolated with glutathione agarose. Precipitated pro-
teins and lysates were separated by 10% SDS-PAGE and blotted with HA and GST antibodies. An asterisk denotes a nonspecific band. In the
right panels, COS7 cells were cotransfected with Flag-tagged RASSF1A and different deletion constructs of GFP-tagged WW45. Cell extracts
were incubated with anti-Flag affinity gel, subjected to 15% SDS-PAGE, and blotted with antibodies as indicated.
commercial sources: anti-RASSF1A (used for western blotting In
Figure 1C) was from Abcam; anti-WW45 (#2953, used for western
blotting in Figure 1C) was from Eric W. McIntush at Bethyl Laborato-
ries; anti-MST2 was from Santa Cruz and Cell Signaling; anti-LATS1
Covance; anti-GST was from Amersham Biosciences; anti-Flag M2
was from Sigma; and anti-g-tubulin was from Sigma. EZview Red
Glutathione affinity gel and EZview Red anti-Flag M2 affinity gel
were from Sigma. The P-MST antibody was kindly provided by
Cell Culture, Transfection, Immunoprecipitation,
and Kinase Assays
HeLa, 293T, and COS7 cells were grown in DMEM (Invitrogen) sup-
plemented with 10% fetal bovine serum. COS7 or 293T cells were
transiently transfected with Fugene 6 (Roche) in 100 mm plates.
For immunoprecipitation, cells were scraped into TNE lysis buffer
(10 mM Tris-HCl [pH 8.0]; 1% NP40; 150 mM NaCl; and 1 mM EDTA)
supplemented with protease inhibitor cocktail (Roche) (20 mM
b-glycerolphosphate, 50 mM sodium fluoride, and 1 mM Na3VO4).
After 30 min on a rotator at 4?C, lysates were collected by centrifu-
gation at 12,200 3 g for 20 min at 4?C. Cell lysates were incubated
with 30 ml EZview Red Glutathione affinity gel or EZview Red anti-
FLAG M2 affinity gel (Sigma) at 4?C on a rotator overnight; alterna-
tively, they were incubated with 4 mg HA antibody plus 30 ml protein
A/G agarose beads (Santa Cruz) under the same conditions. For en-
dogenous coimmunoprecipitation, HeLa cell lysates were prepared
as above but incubated on a rotator overnight at 4?C with 30 ml pro-
tein A/G agarose beads (Santa Cruz) plus one of the following: goat
anti-MST2 antibody (Santa Cruz), rabbit anti-WW45 #4 antiserum, or
rabbit anti-RASSF1A antibody (M304).Precipitated proteins and cell
lysates were subjected to 8% SDS-PAGE followed by western blot-
ting with mouse anti-RASSF1A (Abcam), rabbit anti-WW45 (Bethyl
Figure S2. Binding-Domain Mapping of WW45 on RASSF1A
A diagram of the human RASSF1A protein and its domains is shown at the top. Different deletion constructs of Flag-RASSF1A and HA-WW45
by 10% SDS-PAGE and blotted with the indicated antibodies. Asterisks denote nonspecific bands. Because a Flag-tagged SARAH domain
(amino acids 289–340) of RASSF1A was expressed at very low levels only, we used GST-tagged RASSF1A-289–340 to demonstrate binding
to HA-WW45 (rightmost panels).
Laboratories), goat anti-LATS1 (Santa Cruz), or rabbit anti-MST2
For kinase assays, 293T cell lysates were incubated with 4 mg HA
antibody (Covance) and 30 ml protein A/G agarose beads at 4?C on
a rotator overnight. Beads were washed three times with TNE lysis
buffer and then washed a further three times with kinase buffer
(25 mM HEPES [pH 7.4]; 50 mM NaCl; 5 mM MgCl2; 5 mM MnCl2,
5 mM b-glycerophosphate; and 1 mM dithiothreitol). Kinase reac-
tions were performed at 30?C in kinase buffer containing 10 mM
ATP and 2 mCi [g-32P] ATP for 30 min. Adding SDS loading buffer
and boiling for 10 min stopped the reactions. Samples were ana-
lyzed by SDS-PAGE and autoradiography.
HeLa cells were grown in six-well plates containing coverslips and
fixed with cold methanol for 5 min at 220?C. Rabbit anti-RASSF1A
(M304), rabbit anti-WW45 (#4), goat anti-MST2 (Santa Cruz), rabbit
anti-LATS1 (Novus Biologicals), and mouse anti-g-tubulin (Sigma)
Figure S3. Verification of the RASSF1A and WW45 Antibodies
(A)RASSF1Aantibody. Leftpanels:westernblotswereperformedwith20mgofcelllysates fromdifferentcelllinesandRASSF1Aantibody M304.
As positive and negative controls, COS7 cells were transfected with 3Flag-RASSF1A and 3Flag-RASSF1C, respectively. RASSF1A is not ex-
pressed because of epigenetic silencing in the 293T cell line and in the KRAS-transformed prostate epithelial cell line 267B1. Right panels:
the centrosomal staining for RASSF1A in HeLa cells is lost when the anti-RASSF1A antibody (M304) is preincubated with the peptide used
(B) WW45 antibody. Left panel: a western blot was performed with HeLa cell lysates and anti-WW45 antibody #4. HeLa cells were transfected
with 30 nM of double-stranded siRNA duplexes targeting either luciferase mRNA (as a control) or WW45 mRNA. The intensity of the WW45 band
was reduced in anti-WW45-siRNA-transfected cells. An asterisk denotes a nonspecific band. Right panel: The centrosomal staining for WW45 in
HeLa cells is lost when the WW45 antibody (#4) is preincubated with the peptide used for immunization.
were used as primary antibodies. Goat anti-rabbit IgG Alexa Fluor
488, goat anti-mouse IgG Alexa Fluor 568, donkey anti-goat IgG
Rho, and rabbit anti-mouse Alexa Fluor 488 were used as secondary
antibodies. Images were obtained with an Olympus IX81 micro-
scope and processed in Adobe Photoshop CS.
Time-Lapse Video Microscopy
mice [S2] were prepared from 13.5 dpc (days post-coitum) embryos
and used at passage 3. Asynchronous fibroblasts were imaged by
time-lapse microscopy (NIKONECLIPSE TS100) during mitotic pro-
gression from prometaphase (time 0 when cells began to change
morphology) until cytokinesis was completed. For the generation
of time-lapse series, photographs were collected every 5 min with
of an animation movie, a series of still images (in TIFF format) was
combined with QuickTime 7 Pro software.
Lentiviral Production and Infection
The cDNAs of RASSF1A, WW45, and MST2 were cloned into the len-
tiviral vector pHIV7-CMV-GFP [S3] for the creation of fusion proteins
with GFP at the N terminus. pHIV7-CMV-GFP, pHIV7-CMV-GFP-
RASSF1A, pHIV7-CMV-GFP-WW45, and pHIV7-CMV-GFP-MST2
each were cotransfected with pCMV-G, pCMV-Rev2, and pCHGP-
2 into 293T cells. Forty-eight hours later, the supernatant was col-
lected and passed through a 0.45 mM filter. The virus preparation
was used for infecting Rassf1a2/2MEF cells at passage 2. Forty-
eight hours after infection, the plates were monitored under the
video microscope. Flow cytometry was performed so that the per-
centage of GFP-positive cells could be confirmed as being >90%.
S1. Liu, L., Tommasi, S., Lee, D.H., Dammann, R., and Pfeifer, G.P.
(2003). Control of microtubule stability by the RASSF1A tumor
suppressor. Oncogene 22, 8125–8136.
S2. Tommasi, S., Dammann, R., Zhang, Z., Wang, Y., Liu, L., Tsark,
W.M., Wilczynski, S.P., Li, J., You, M., and Pfeifer, G.P. (2005).
Tumor susceptibility of Rassf1a knockout mice. Cancer Res.
S3. Yam, P.Y., Li, S., Wu, J., Hu, J., Zaia, J.A., and Yee, J.K. (2002).
Design of HIV vectors for efficient gene delivery into human
hematopoietic cells. Mol. Ther. 5, 479–484.
Figure S4. Anaphase Location of RASSF1A,
WW45, MST2, and LATS1
HeLa cells were grown on coverslips in six-
well plates, fixed, and stained with specific
antibodies. Cells in anaphase are shown.
Purified rabbit anti-RASSF1A, rabbit anti-
WW45, goat anti-MST2, or rabbit anti-
LATS1 antibodies were used for costaining
Figure S5. Colocalization of MST2 with RASSF1A, WW45, and LATS1
HeLa cells were grown on coverslips in six-well plates, fixed, and stained with specific antibodies. Cells in interphase (prophase for RASSF1A)
and metaphase are shown. Purified goat anti-MST2 together with rabbit anti-LATS1 (A), rabbit anti-WW45 (B), or rabbit anti-RASSF1A (C) was
used for costaining with DAPI.