(Received for publication, October 10, 1995; and in revised form, November 7, 1995)
From the
Ras (Ha-Ras, Ki-Ras, N-Ras) is implicated in the regulation of
various cell functions such as gene expression and cell proliferation
downstream from specific extracellular signals. Here, we partially
purified a Ras-interacting protein with molecular mass of about 180 kDa
(p180) from bovine brain membrane extract by glutathione S-transferase (GST)-Ha-Ras affinity column chromatography.
This protein bound to the GTPS (guanosine
5`-(3-O-thio)triphosphate, a nonhydrolyzable GTP
analog)
GST-Ha-Ras affinity column but not to those containing
GDP
GST-Ha-Ras or GTP
S
GST-Ha-Ras with a mutation in the
effector domain (Ha-Ras
). The amino acid sequences of the
peptides derived from p180 were almost identical to those of human AF-6
that is identified as the fusion partner of the ALL-1 protein. The
ALL-1/AF-6 chimeric protein is the critical product of the t (6:11)
abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg
homology repeat (DHR) motif and shows a high degree of sequence
similarity with Drosophila Canoe, which is assumed to function
downstream from Notch in a common developmental pathway. The
recombinant N-terminal domain of AF-6 and Canoe specifically interacted
with GTP
S
GST-Ha-Ras. The known Ras target c-Raf-1 inhibited
the interaction of AF-6 with GTP
S
GST-Ha-Ras. These results
indicate that AF-6 and Canoe are putative targets for Ras.
Ras (Ha-Ras, Ki-Ras, N-Ras) is a signal-transducing guanine
nucleotide-binding protein for tyrosine kinase-type receptors such as
epidermal growth factor receptors and the Src family, leading to a
mitogenic response and differentiation (for reviews, see Refs. 1 and
2). Ras has GDP-bound inactive and GTP-bound active forms, the latter
of which makes physical contact with targets. Intensive investigations
revealed that the Raf kinase family, consisting of c-Raf-1 (for
reviews, see (3) and (4) ), A-Raf(5) , and
B-Raf(6, 7, 8, 9) , is one of the
direct targets for Ras. The activated Raf phosphorylates MAP ()kinase kinase and activates it. Consequently the activated
MAP kinase kinase activates MAP kinase, leading to the expression of
certain genes such as c-fos (for reviews, see (10) and (11) ). Several molecules interacting with
activated Ras in addition to Raf have been identified in mammals. These
include phosphatidylinositol-3-OH kinase(12) , Ral
GDS(13, 14) , and Rin1(15) . On the basis of
these observations, a variety of Ras targets may account for the
pleiotropic functions of Ras. To understand the molecular mechanism of
pleiotropic functions of Ras, it is essential to identify novel targets
for Ras.
In the present study, we discovered and partially purified another putative target for Ras with a molecular mass of about 180 kDa (p180) by use of GST-Ha-Ras affinity column chromatography and identified it as AF-6(16) , whose structure resembles that of Drosophila Canoe, which is involved in the Notch signaling pathway(17) .
To detect molecules interacting with Ha-Ras, the bovine brain
membrane extract was loaded onto GST-Ha-Ras affinity columns. The
proteins bound to the affinity columns were coeluted with GST-Ha-Ras by
addition of glutathione. Proteins with a molecular mass of about 180
kDa (p180) and 195 kDa (p195) were detected in the glutathione eluate
from the GTPS
GST-Ha-Ras affinity column but not from those
containing GST or GDP
GST-Ha-Ras (Fig. 1). Neither p180 nor
p195 was detected in the eluate of the affinity column for
GTP
S
GST-Ha-Ras
, which has a mutation in the
effector-interacting domain(1, 2) . We further
confirmed the specificity of the interaction by affinity column
chromatography using GST-R-Ras, GST-RalA, and GST-RhoA. Less p180 and
p195 were eluted from the GTP
S
GST-R-Ras affinity column but
not from the GDP
GST-R-Ras affinity column (data not shown).
Neither p180 nor p195 was eluted from the GST-RalA or GST-RhoA affinity
column (data not shown).
Figure 1:
Purification of Ha-Ras-interacting
proteins. The membrane extract was loaded onto a glutathione-Sepharose
4B column containing either GST, GDPGST-Ha-Ras,
GTP
S
GST-Ha-Ras, or GTP
S
GST-Ha-Ras
.
Bound proteins were coeluted with the respective GST-fusion proteins by
addition of glutathione. Aliquots (40 µl each) from the glutathione
eluates were resolved by SDS-PAGE followed by silver staining. Lane
1, GST; lane 2, GDP
GST-Ha-Ras; lane 3,
GTP
S
GST-Ha-Ras; lane 4,
GTP
S
GST-Ha-Ras
. The arrow and arrowhead denote the positions of p180 and p195, respectively.
The results are representative of three independent
experiments.
To identify the
GTPS
Ha-Ras-interacting molecule, p180 was subjected to amino
acid sequencing as described under ``Experimental
Procedures.'' Six peptide sequences derived from p180 were
determined. These were: 1) STATTQDVLE; 2) DMPETSFTR; 3) LPYLVELSPDG; 4)
PGIVQETTFDLG; 5) YAPDDIPNINS; and 6) LLLEWQFQK. All six peptide
sequences were almost identical to the deduced amino acid sequence of
human AF-6, which is the fusion partner of the ALL-1
protein(16) . The ALL-1/AF-6 chimeric protein is the critical
product of the t (6:11) abnormality associated with some human
leukemia. Furthermore, p180 was recognized by the antibody raised
against human AF-6 (Fig. 2). The calculated molecular mass of
human AF-6 is 181,777 Da, which is close to the apparent molecular mass
of p180 estimated by SDS-PAGE. We therefore concluded that p180 is the
bovine counterpart of human AF-6 and hereafter referred to it as AF-6.
Since this antibody cross-reacted with p195 weakly (data not shown),
p195 may be an isoform or an alternatively spliced form of AF-6.
Figure 2: Immunoblot analysis of p180. Protein p180 was immunoblotted against the anti-AF-6 antibody. Lane 1, with preimmune serum; lane 2, with the anti-AF-6 antibody. The arrow denotes the position of p180. The results are representative of three independent experiments.
To
address whether or not recombinant AF-6 interacts with
GTPS
Ha-Ras, GST-small G proteins immobilized on beads were
mixed with the in vitro translated N-terminal domain of AF-6
(36-848 aa), and interacting proteins were coeluted with
GST-small G proteins by the addition of glutathione. The in vitro translated AF-6 (36-848 aa) was coeluted with
GTP
S
GST-Ha-Ras but weakly with GDP
GST-Ha-Ras,
GTP
S
GST-Ha-Ras
, GST-R-Ras, GST-RalA, and
GST-RhoA (Fig. 3a). The band with
GTP
S
GST-Ha-Ras
was very faint (lane
5), and the bands with GDP
GST-RalA and
GTP
S
GST-RalA (lanes 8 and 9) were a little
bit stronger than those with GDP
GST-R-Ras and
GTP
S
GST-R-Ras (lanes 6 and 7). The weak
bands detected in the eluates other than that from
GTP
S
GST-Ha-Ras may result from the weak interaction of AF-6
with the respective small G proteins. Although some AF-6 in the
membrane extract was slightly retained on the GTP
S
GST-R-Ras
affinity column (data not shown), the in vitro translated AF-6
was not. This may be due to the lower affinity of AF-6 for
GTP
S
GST-R-Ras than that for GTP
S
GST-Ha-Ras. To
determine the Ras-interacting domain of AF-6 more accurately, a similar
experiment was performed using the shorter N-terminal domain of AF-6
(36-206 aa). A similar retention was observed when the shorter
N-terminal domain of AF-6 (36-206 aa) was employed (Fig. 3b).
Figure 3:
Interaction of AF-6 with activated Ha-Ras. a, the in vitro translated AF-6 (36-848 aa) was
mixed with GST-small G proteins immobilized to glutathione-Sepharose 4B
beads. The interacting proteins were coeluted with GST-small G proteins
by addition of glutathione. Aliquots (40 µl) of the eluates were
subjected to SDS-PAGE and vacuum-dried followed by autoradiography. Lane 1, in vitro translated AF-6; lane 2,
GST; lane 3, GDPGST-Ha-Ras; lane 4,
GTP
S
GST-Ha-Ras; lane 5,
GTP
S
GST-Ha-Ras
; lane 6,
GDP
GST-R-Ras; lane 7, GTP
S
GST-R-Ras; lane
8, GDP
GST-RalA; lane 9, GTP
S
GST-RalA; lane 10, GDP
GST-RhoA; lane 11,
GTP
S
GST-RhoA. The arrow denotes the position of
AF-6. b, the in vitro translated AF-6 (36-206
aa) or Canoe (1-217 aa) was mixed with glutathione-Sepharose 4B
beads containing GST-Ha-Ras. Lane 1, in vitro translated AF-6; lane 2, GST; lane 3,
GDP
GST-Ha-Ras; lane 4, GTP
S
GST-Ha-Ras; lane 5, GTP
S
GST-Ha-Ras
; lane
6, in vitro translated Canoe; lane 7, GST; lane 8, GDP
GST-Ha-Ras; lane 9,
GTP
S
GST-Ha-Ras; lane 10,
GTP
S
GST-Ha-Ras
. The arrow and arrowhead denote the positions of AF-6 and Canoe,
respectively. c, MBP-AF-6 was mixed with GST-Ha-Ras affinity
beads. Lane 1, GST; lane 2, GDP
GST-Ha-Ras; lane 3, GTP
S
GST-Ha-Ras; lane 4,
GTP
S
GST-Ha-Ras
; lane 5,
GTP
S
GST-Ha-Ras with MBP-c-Raf-1. The results are
representative of three independent
experiments.
A homology search of the GenBank protein
data base revealed a high degree of sequence similarity of AF-6 with Drosophila Canoe (Fig. 4), which is assumed to function
downstream from Notch in a common developmental pathway(17) .
Since Canoe was presumed to interact with Ras in the same manner as
AF-6, the interaction of Canoe was investigated. The in vitro translated N-terminal domain of Canoe (1-217 aa) was also
coeluted with GTPS
GST-Ha-Ras and scarcely with GST,
GDP
GST-Ha-Ras, and GTP
S
GST-Ha-Ras
(Fig. 3b).
Figure 4: Schematic representation of AF-6 and Canoe structures. The numbers indicate the amino acid sequence identities in each domain. DHR, Dlg homology repeat.
We examined whether or not AF-6
directly interacts with GTPS
Ha-Ras. The shorter N-terminal
domain of AF-6 (36-206 aa) was expressed as an MBP fusion protein
(MBP-AF-6) and mixed with immobilized GST-Ha-Ras. Interacting proteins
were coeluted with GST-Ha-Ras by the addition of glutathione. MBP-AF-6
was coeluted with GTP
S
GST-Ha-Ras but not with GST,
GDP
GST-Ha-Ras, or GTP
S
GST-Ha-Ras
(Fig. 3c). The band corresponding to molecular
mass of about 55 kDa may be a degraded product of MBP-AF-6.
The
apparent K values for MBP-AF-6 and MBP-c-Raf-1
were estimated to be about 250 and 200 nM, respectively, under
the conditions (data not shown). Since c-Raf-1 interacts with activated
Ras via the effector domain(1, 2) , we examined
whether or not c-Raf-1 competes with AF-6 for interaction with
activated Ha-Ras. An excess amount of MBP-c-Raf-1 inhibited the
interaction of the MBP-AF-6 with GTP
S
GST-Ha-Ras (Fig. 3c).
In this study, we purified a Ras-interacting protein (p180) from a bovine brain membrane extract. We identified it as AF-6, which has a GLGF/DHR motif and shows a high degree of sequence similarity with Drosophila Canoe(16, 17) . The recombinant AF-6 and Canoe specifically interacted with activated Ha-Ras. Furthermore, c-Raf-1 inhibited the interaction of AF-6 with activated Ha-Ras. These results indicate that AF-6 and Canoe serve as putative targets for Ras.
We
showed that activated Ras interacted with the N-terminal domains of
AF-6 and Canoe. These domains show a high degree of sequence similarity
to each other, indicating that this unique domain confers specificity
for the GTPRas complex. The direct interaction of c-Raf-1, A-Raf,
B-Raf, phosphatidylinositol-3-OH kinase, Ral GDS, and Rin1 with
activated Ras has been
demonstrated(3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15) .
The Ras-interacting interfaces of these proteins have been determined.
There is no obvious homology among Ras-interacting interfaces of
c-Raf-1, phosphatidylinositol-3-OH kinase, Ral GDS, Rin1, and
AF-6/Canoe, indicating that activated Ras can recognize a variety of
target interfaces. This diversity of Ras-interacting interfaces may
allow a range of downstream pathways from Ras to induce appropriate
cellular responses to extracellular signals.
AF-6 and Canoe are homologous to each other and share a common domain organization (Fig. 4)(16, 17) . The most highly conserved region among them is a GLGF/DHR motif, which is found in a number of other proteins including Drosophila discs-large tumor suppressor gene product (Dlg)(24) , dishevelled gene product (25, 26) , an intracellular protein-tyrosine phosphatase (PTP-meg)(27) , postsynaptic density protein 95 (PSD-95)(28) , and a tight junction-associated protein ZO-1(29, 30) . The GLGF/DHR motif is thought to function to localize them at the specialized sites of cell-cell contact by forming a complex with specific proteins such as protein 4.1 homologues(31) . The structural feature of AF-6 and Canoe suggests that they locate at the junction of plasma membrane and cytoskeleton, where they may regulate signal transduction and cytoskeleton.
The N terminus of AF-6 flanked by the GLGF/DHR motif also shares a high homology with that of Canoe, to both of which activated Ras binds specifically. Canoe has been postulated to function downstream from Notch and to mediate interactions between the Notch cascade and other signaling pathways(17) . Although AF-6 function remains obscure, the similar structural feature and property of AF-6 and Canoe suggest that the AF-6/Canoe family may serve as an intracellular signaling component controlled by two distinct signaling pathways such as Ras and Notch. Our preliminary experiments suggest that Canoe is genetically linked to Ras1 in Drosophila eye development. Further studies are required to understand the roles of AF-6/Canoe family in signal transduction.