From the Transcription Laboratory, Imperial Cancer
Research Fund Laboratories, 44 Lincoln's Inn Fields,
London WC2A 3PX and
Division of Yeast Genetics, National
Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, United Kingdom
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ABSTRACT |
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To identify potential RhoA effector proteins, we
conducted a two-hybrid screen for cDNAs encoding proteins that
interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA
effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a
p140 mDia-related protein), and the guanine nucleotide exchange factor,
mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both
RhoA and Cdc42 in a GTP-dependent manner in
vitro. Comparison of the ROCK-I and Kinectin sequences revealed a
short region of sequence homology that is both required for interaction
in the two-hybrid assay and sufficient for weak interaction in
vitro. Sequences related to the ROCK-I/Kinectin sequence homology
are present in heterotrimeric G protein subunits and in the
Saccharomyces cerevisiae Skn7 protein. We show that
2
and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1,
both in vivo and in vitro. Functional assays in
yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required
for its function in vivo.
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INTRODUCTION |
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Members of the Rho family of GTPases regulate diverse cellular
processes ranging from cytoskeletal organization to gene expression and
cell transformation. Upon binding GTP, these Ras-like proteins interact
with effector proteins to induce downstream signals (for reviews see
Refs. 1-3). Recent biochemical and genetic studies have identified
many potential Rho effectors in both mammalian cells and the budding
yeast Saccharomyces cerevisiae. Mammalian RhoA interacts
with members of the PKN/PRK and ROCK/ROK protein kinase families
(4-10). The ROCKs are clearly involved in cytoskeletal rearrangements
(6, 11-13), but the functions of the PKN/PRK kinases remain obscure.
RhoA also interacts with several apparently non-catalytic effectors
including Rhophilin, Rhotekin, Citron, the myosin-binding subunit of
myosin light chain phosphatase
(MBS),1 p140 mDia, Kinectin,
and p116RIP (9-11, 14-18). RhoA interactions with MBS, p140 mDia, and
Kinectin are likely to be involved in contractile events, actin
polymerization and cytokinesis, and motility, respectively (11, 17, 19,
20). In S. cerevisiae, RHO1, an essential gene
(21, 22), controls activity of 1,3--glucan synthase, the enzyme that
synthesizes cell wall glucan polymers (23, 24), and BNI1, a
gene involved in cytoskeletal events and cytokinesis (25, 26). Rho1
regulates the PKC1-MPK1 MAP kinase pathway that controls
cell wall integrity (27, 28) and the Rlm1 transcription factor (29,
30). Interestingly, Skn7, a yeast two-component protein, shows genetic
interactions with the PKC1 pathway and exhibits several
properties that suggest that it too may be a Rho1 effector
(31-33).
Sequence elements involved in the interaction between Rho family
GTPases and their effectors are of considerable interest since their
definition should permit the identification of further potential
effector proteins. The first such motif to be identified was the CRIB
(Cdc42/Rac interactive binding) motif, which specifies interaction with
Rac1 and Cdc42, but not RhoA, and has the consensus sequence
ISXPX2 or 3FXHX2(H/T)(V/A)(G/D/Q)
(34). A second motif, REM-1 (Rho effector motif class 1), is found in the N-terminal Rho-binding domains of the RhoA effectors PKN/PRK1, PRK2, Rhotekin, and Rhophilin and has the consensus
(L/I/F)X2(E/K)X2(V/I/L)X2GX(E/K/R)(N/R/Q) (9, 14). REM-1 appears specific for RhoA, although PRK2 has also been
reported to bind Rac1 (8). Although the REM-1 motif is reiterated three
times at the N terminus of the PKN/PRK kinases, only a single copy is
found in Rhophilin and Rhotekin. REM-1 motifs appear to differ in their
ability to discriminate between the GTP- and GDP-bound forms of
RhoA.2 No sequence motifs
common to other RhoA effectors have been identified, although the
defined Rho-binding regions of Citron, Kinectin, and the ROCKs are all
found in regions of extended -helical coiled-coil structure.
To identify potential RhoA effectors, we conducted a two-hybrid screen
with RhoA.V14 as a bait. In addition to ROCK-I, we identified cDNAs
encoding Kinectin, a p140 mDia-related protein, and the mouse homolog
of the NET1 GEF. Comparison of the ROCK-I and Kinectin sequences
revealed a short region of sequence homology that is both required for
and sufficient for interaction with RhoA and Cdc42. A similar sequence
is found in both heterotrimeric G protein subunits and the yeast
Skn7 protein and is also required for the interaction of these proteins
with RhoA. In addition to defining a RhoA interaction domain, the
ROCK-I/Kinectin homology region is required for the function of Skn7
in vivo, consistent with the notion that Skn7 represents
a novel Rho effector.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
Plasmids were constructed by standard techniques;
details are available on request. The SKN7
(GenBankTM accession number U00485; Ref. 31) and human 2
subunit (GenBankTM accession number M16538; Ref. 35) coding
sequences were obtained by PCR and their DNA sequences confirmed.
GAL4 DNA-binding Domain Fusions-- Activated 9E10-tagged RhoA and Cdc42 were subcloned from mammalian expression plasmids (36) as NcoI + Xho fragments into pGBT9; the RhoA CAAX motif was inactivated by a C190S mutation introduced by PCR and that of Cdc42 by truncation generating Cdc42-(1-178)-ID. Yeast Rho1 was isolated using PCR and Rho1.G19V/C206S constructed by standard techniques.
GAL4 Activation Domain Fusions--
cDNA clones isolated in
the two-hybrid screen are summarized in Table I. For further two-hybrid
analyses DNA fragments were inserted into derivatives of pGAD424 and
pGAD10 (CLONTECH). A cDNA encoding a CRIB
domain protein related to MSE55 (34) was used as a specificity control
in the two-hybrid assay.3 The
plasmids encode the following sequences C-terminal to the Gal4
activation domain. cDNA clone D1 encodes IWNSDPREFT(ROCK-I codons 300-1030)-KKKVNSRDL. cDNA clone D4 encodes
IWNSDPRNLPSSP-(ROCK-I codons 456-1028)-VNLERSMNRRY. cDNA clone D9
encodes IWNSDPREFT-(ROCK-I codons 349-1025)-GELERSMNRRY.
GAD-ROCK-(831-1010) encodes IEFPM-(ROCK-I codons
831-1010)-DLQRFMNRRY. GAD-ROCK-(831-1010)TT is the same as
GAD-ROCK-(831-1010) but with K1005T/L1006T.
GADROCK-(831-1010)-HR encodes ISRGS-(ROCK-I codons
831-1010)-FQIYES with codons 950-966 deleted. GAD-ROCK.HR encodes
ISRGS-(ROCK-I codons 950-972)-FQIYES. GAD-Kinectin-(1053-1327) (cDNA clones D2 and D3) encodes
IWNSDPRDLP-(Kinectin codons 1053-1327).
GAD-Kinectin-(1053-1327)-
HR encodes IEFPMGRDLP-(Kinectin codons
1053-1327) with codons 1191-1215 replaced by GS. GAD-mDia2-(47-257) (cDNA clone D7) encodes mDia2 IWNSDPREF-(mDia2 codons
47-800)-VNSREIYES. GAD-NET (clone D5) encodes IWNSDPRDLP-(15 codons
from mNET1 5'UT)-(mNET1 1-595). GAD-SKN7 encodes
ISGRS-(Skn7 codons 1-623). GAD-SKN7
HR encodes IEF (Skn7
codons 1-623) with codons 237-260 replaced by G. GAD-
2.WT encodes
ISRGS-(Human
2 codons 1-340). GAD-
2
HR encodes IEFPM-(
2
codons 24-340). GAD-
2HR encodes ISRGS-(
2 codons 1-32)-LEIPDL.
GAD-PKN.N encodes IEFPM (PKN codons 1-511). GAD-PAK.N encodes
IEFPMAGS-(Rat PAK
codons 1-252)-RRPAEIYES.
GST Fusion Proteins--
All fusion proteins were made using
pGEX-KG and encode the following sequences. pGEX-RhoA.WT and
pGEX-Cdc42WT encode wild type RhoA and Cdc42, respectively.
GST-ROCK-(831-1010) encodes (ROCK-I codons 831-1010)-LELKLNSS.
GST-ROCK-(831-1010)TT is the same as GST-ROCK-(831-1010) with
K1005T/L1006T. GST-ROCK-(831-1010)-HR encodes ROCK-(831-1010)-ES,
with codons 950-966 deleted. GST-ROCK.HR encodes ROCK-(950-972)-F.
GST-Kinectin-(1053-1327) encodes Kinectin codons 1053-1372.
GST-Kinectin-(1053-1327)-
HR is GST-Kinectin-(1053-1327) with
codons 1191-1215 replaced by GS. GST-mDia2-(47-257) encodes (mDia2
codons 47-257)-QLNSS. GST-NET encodes mNET1 (codons 122-595). GST-SKN7 encodes Skn7 1-623 from Lee Johnston.
GST-SKN7
HR encodes (Skn7 codons 1-623) with codons
237-260 replaced by G. GST-
2.WT encodes (
2 codons 1-340).
GST-
2
HR encodes (
2 codons 24-340). GST-
2HR encodes (
2
codons 1-32)-LELKLNSS. GST-PAK.N encodes (Rat PAK
codons
1-252)-EIRRLELKLNSS. GST-PKN.N encodes (PKN codons 1-511).
Two-hybrid Screen and Yeast Manipulations-- Yeast strains and manipulations were as described previously (33, 37, 38). For the two-hybrid screen, yeast strain HF7c carrying pGBT9-RhoA.V14/S190 was used in conjunction with a mouse T-helper cell cDNA library in pSE1107 (39). 4 million tranformants were plated onto selective plates lacking histidine and grown for 3 days at 30 °C. Colonies were rescreened for expression of the lacZ marker after lifting onto nitrocellulose filters (38).
Recombinant Proteins-- Overnight cultures were diluted 1:10 to 50 ml, grown for 3 h, then lysed by sonication in 5 ml of RB (100 mM NaCl, 5 mM MgCl2, 25 mM Tris, pH 7.2) with protease inhibitors, adsorbed to 0.5 ml of glutathione-Sepharose 4B beads, and washed extensively. RhoA.WT-9E10 and Cdc42.WT-9E10 proteins were released by overnight incubation with thrombin (5 units; Sigma) at 4 °C in RB; thrombin was removed by adsorption to p-aminobenzamidine-Sepharose 6B (0.5 ml; Sigma). Protein concentrations were determined by dye-binding assay (Bio-Rad) or by comparison to known standards on Coomassie-stained SDS-polyacrylamide gels.
In Vitro Binding Assay--
Equimolar amounts of each GST-fusion
protein (~100-300 ng) were bound to glutathione-Sepharose beads and
incubated with 10 ng of GTPS- or GDP-loaded RhoA.WT-9E10 or
Cdc42.WT-9E10 at 4 °C for 2 h, with agitation, in RB containing
0.5 mg/ml bovine serum albumin. The beads were them washed in ice-cold
RB, 0.1% Nonidet P-40, and bound GTPase was eluted by boiling in
SDS-PAGE sample buffer. Following fractionation by SDS-PAGE,
GTPases were detected by immunoblotting with the 9E10
antibody.
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RESULTS |
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Identification of Potential RhoA Effectors by the Two-hybrid Screen-- We performed a two-hybrid screen for proteins that can interact with the activated form of RhoA. A fusion gene, Gal4-RhoA.V14/S190, was constructed in which the Gal4 DNA-binding domain is fused N-terminal to activated human RhoA (RhoA.V14), carrying an additional mutation at its C terminus to destroy the CAAX motif. Yeast HF7c cells expressing Gal4-RhoA.V14/S190 were used to screen a library of Gal4 activation domain-tagged mouse T-cell cDNA. Seven transformants exhibited Gal4-RhoA.V14/S190-dependent activation of both the HIS3 and LacZ markers in HF7c cells. The cDNAs were characterized by partial DNA sequencing (Table I). Plasmids D1, D4, and D9 carry partial cDNAs encoding the RhoA effector kinase ROCK-I (p160ROCK; Ref. 7). Plasmids D2 and D3 carried the same partial Kinectin cDNA (20); interestingly, although Kinectin was previously identified as a putative RhoA effector, the cDNA fragment isolated in our screen does not overlap that isolated in the previous screen (15; see "Discussion"). Plasmid D5 carried a complete open reading frame 83% identical to the putative Rho family guanine nucleotide exchange factor NET1 (40); functional characterization of this protein will be published elsewhere.4 Plasmid D7 contains a cDNA related to p140 mDia, as identified in a previous screen for RhoA effector proteins (17) which is related to the Drosophila gene Diaphanous (41). In vitro experiments described below indicated that the RhoA-binding domain of this protein is located in its N-terminal sequences; we therefore sequenced this region, which spans codons 47-257, and compared it with p140 mDia and with Drosophila Diaphanous (Fig. 1). A full characterization of this protein, to which we refer as mDia2, is currently in progress.
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RhoA and Cdc42 Bind to Kinectin, ROCK-I, mDia-2 in a GTP-dependent Manner-- We used the two-hybrid assay to investigate the interactions between RhoA, its yeast homolog Rho1, and human Cdc42 and the proteins identified in the screen, in each case using Gal4-GTPase fusion proteins containing activating mutations and mutated CAAX motifs. Each of the three GTPases could interact with all the proteins in the assay (Fig. 2A). To confirm the specificity of the interactions with Cdc42, we examined interaction of an MSE55-related protein isolated in a screen for Cdc42 effectors5; this CRIB motif-containing protein interacted with Cdc42.V12, but not RhoA.V14, in the two-hybrid assay in agreement with previous results (Fig. 2A; Ref. 34).
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mROCK-I and Kinectin Have Similarities within Their Rho-binding
Domains--
The RhoA effectors Rhotekin, Rhophilin, and PKN share a
region of homology within their Rho-binding domains (the REM-1 motif; 14). Previous studies of the ROCK proteins have defined a short region
within its C-terminal coiled-coiled region that suffices for
interaction with RhoA (4, 5, 43). We therefore compared the sequence
spanning this region with the sequences of Kinectin, mDia2, and mNET1
to identify potential Rho-binding sequence motifs. Although the maximum
homology between the ROCK proteins corresponds to ROCK-I residues
995-1014 (5, 7) a region of substantial similarity between ROCK-I and
Kinectin is found N-terminal to this, corresponding to ROCK-I residues
950-972 (Fig. 3A). This region lies within the "leucine zipper" region of ROCK-I and is also within a coiled-coil region in Kinectin. No substantial sequence homology was found with mDia2 (data not shown). We used the homology between ROCK-I and Kinectin to screen the sequence data bases for
similar sequences using the Blastp program (NCBI). Among many coiled-coil proteins identified, this search detected a second Kinectin
sequence element N-terminal to that in our cDNA clone (residues
832-854) and a region at the N terminus of heterotrimeric G protein
subunits. We also observed that the sequence of the S. cerevisiae SKN7 gene, which interacts genetically with the RHO1-PKC1 pathway (31-33), also contains a
region of similarity to the ROCK-I/Kinectin homology, again within a
region predicted to form a coiled-coil structure (Fig. 3B;
see "Discussion"). These sequences are compared in Fig.
3B.
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The ROCK-I/Kinectin Homology Is Required for Interaction with Rho
Proteins--
To investigate the significance of the ROCK-I/Kinectin
homology, we examined its role in the interactions with RhoA and Cdc42 using both two-hybrid assays (Fig. 4) and
in vitro biochemical assays (Fig.
5). To facilitate mutagenesis of ROCK-I,
we examined a shorter ROCK-I fragment containing codons 831-1010,
which interacts strongly with RhoA.V14 and Cdc42.V12 in the two-hybrid
assay (Fig. 4A, rows 1 and 2) and with GTP-loaded
RhoA and Cdc42 in the in vitro binding assay (Fig. 5A,
lanes 5). Sequences encompassing the homology were deleted from
both ROCK-I and Kinectin, and the interactions of the resulting
proteins with RhoA and Cdc42 were examined. Deletion of the homology
region (codons 950-972) from ROCK-I-(831-1010) generates a protein
that does not interact with RhoA or Cdc42 in either assay
(ROCK-I-(831-1010)-HR: Fig. 4A, row 3; Fig. 5A,
lanes 6). Similarly deletion of the homology region from Kinectin
also abolished interaction with RhoA and Cdc42 in both assays
(Kinectin-(1053-1327)-
HR: Fig. 4A, row 7; Fig. 4B, row 2; Fig. 5A, lanes 3 and 4). The
ROCK-I/Kinectin homology region is therefore required for interaction
of both proteins with RhoA and Cdc42.
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The Homology Region Is Sufficient for Interaction with RhoA or
Cdc42--
We next tested whether the ROCK-I/Kinectin homology region
is sufficient for binding to RhoA and Cdc42. Sequences encompassing the
homology from ROCK (codons 950-972) or 2 (codons 1-32) were inserted into appropriate plasmids for use in the two-hybrid and in vitro interaction assays. In addition to deletion of the
ROCK-I/Kinectin homology region, previous studies have demonstrated
that point mutations of ROK-
(ROCK-II) residues C-terminal to the
homology severely impair its interaction with RhoA in GTPase overlay
assays (5). We therefore constructed an analogous mutant,
ROCK-I-(831-1010)-TT and compared its binding properties with those of
the isolated ROCK-I/Kinectin homology region.
The ROCK-Kinectin Homology Region Is Required for Skn7
Function--
The results presented in the preceding section provide
strong evidence that Skn7 interacts with both RhoA and Rho1 and show that the ROCK-I/Kinectin homology region is required for this interaction. We therefore examined the role of the ROCK-I/Kinectin homology in Skn7 function in yeast using a number of different assays
(Table II). These assays rely either on
measuring the effects of Skn7 overexpression in different genetic
backgrounds or on measuring the ability of different Skn7 mutants to
suppress the effects of SKN7 deletions. High level
overexpression of SKN7 from the GAL1 promoter in
wild type cells is lethal, probably owing to weakening of the cell wall
(33). Skn7 overexpression from high copy number plasmids also activates
the MCB promoter element which is partly responsible for G1
cyclin gene expression; Skn7 overexpression can therefore bypass the
normal requirement for the SWI4 and SWI6 gene
products, allowing growth of swi4ts
swi6 cells at the nonpermissive temperature (33). In addition, Skn7 overexpression partially suppresses the temperature-sensitive phenotype of cells expressing human RhoA, allowing them to grow at
35.5 °C; in contrast, it exacerbates the severity of the
temperature-sensitive cdc42 mutation, preventing growth at
35.5 °C.6 Deletion of
SKN7 has a number of effects, rendering cells acutely sensitive to oxidative stress (44) and preventing growth of pkc1-8 cells at 37 °C (32, 33, 37). Moreover, deletion of SKN7 prevents suppression of the
swi4ts swi6
double mutation by Mbp1
overexpression.7
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DISCUSSION |
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In this work we used a two-hybrid screen to identify potential effector proteins of the mammalian Rho family GTPase RhoA. We identified cDNAs encoding two previously characterized effectors, ROCK-I and Kinectin, together with cDNAs for mNET1, the mouse homolog of NET1 (40), a putative guanine nucleotide exchange factor (GEF), and a novel protein, mDia2, which is related to p140 mDia (17). Our Kinectin cDNA spans codons 1053-1327, a region that is distinct from that identified as a RhoA-binding domain in a previous two-hybrid screen (15), which suggests that the protein contains multiple Rho-binding elements. Although the ROCK-I, Kinectin and mDia2 proteins bound to both RhoA and Cdc42 in a GTP-dependent manner, mNET1 exhibited similar affinities for both GTP- and GDP-bound RhoA. This behavior was not unexpected because the protein contains a Dbl homology domain, associated with Rho family guanine nucleotide exchange factor activity (for review see Ref. 45); indeed, mNET1 acts as a RhoA GEF both in vitro and in vivo.4 In addition to significant sequence homology between mDia2 and p140 mDia within the RhoA-binding domain, mDia2 also contains a region homologous to the p140 mDia formin homology domain; characterization of the mDia2 protein is in progress.
Our data implicate ROCK-I, Kinectin, and mDia2 as effectors for both RhoA and Cdc42. However, although previous studies of ROCK-I using overlay and two-hybrid assays broadly concur concerning its interactions with RhoA, its ability to interact with other Rho family proteins has been contentious. Two studies reported weak interaction between ROCK-I and activated Cdc42, although one observed no interaction with wild type Cdc42 (46, 47); interactions with activated Rac1 have also been reported (47, 48). We are confident that the interaction between wild type Cdc42 and ROCK-I detected by our assays is specific, because specificity controls with the interaction domains of PAK65, an MSE55-related protein, and PKN clearly demonstrate Cdc42/Rac-specific and RhoA-specific interactions by the CRIB and REM-1 interaction domains. We have also observed interaction between GTPase binding fragments of ROCK-I and activated Cdc42.V12 in microinjection assays in mammalian cells.5 The discrepancy between our data and those obtained by others using overlay assays may reflect the stringency of the overlay assay compared with the two-hybrid and affinity chromatography approaches used here.
Sequence comparison of the Kinectin cDNA recovered from the
two-hybrid assay with the minimal RhoA-binding domain of ROCK-I (5, 43)
revealed a 20 amino acid homology between the two proteins. This
sequence, which is unrelated to that of the mDia2 Rho interaction
domain, is both necessary for interaction with GTP-bound RhoA and Cdc42
and can by itself can interact weakly with these GTPases. Intriguingly,
the distinct Kinectin cDNA previously isolated as a potential RhoA
effector also contains a sequence related to the ROCK-I/Kinectin
homology region (15). The ROCK-I/Kinectin homology occurs within a
region of predicted extended coiled-coil structure, and the REM-1 motif
that mediates interactions with PKN/PRK1, Rhotekin, and Rhophilin (14)
also may have helical character.4 Sequences related to the
ROCK-I/Kinectin homology are present in heterotrimeric G protein subunits and in the yeast two-component protein Skn7, also within
putative coiled-coil regions. However, although we could demonstrate
that both
2 and Skn7 interact with GTP-bound RhoA and Cdc42 and that
the interaction is dependent on the ROCK/Kinectin homology, the
strongly helical character of the sequence has precluded its use as a
search string in data base searches for further Rho-interacting
proteins.9
The ability of the isolated ROCK-I/Kinectin homology to bind RhoA weakly in vitro suggests that it makes direct contacts with the GTPase. However, previous studies of ROCK-I have shown that point mutations or deletion of sequences outside the ROCK-I/Kinectin homology also reduce RhoA binding in both two-hybrid and overlay assays, although the precise effects vary, presumably owing to the different assay conditions used (5, 43). In agreement with a previous study (5) we found that mutation of sequences highly conserved between ROCK-I and ROCK-II substantially reduced interaction with RhoA in both our assays. These sequences might act to stabilize the secondary structure of the ROCK-I/Kinectin homology; alternatively, they might represent a second GTPase docking site. Further studies will be necessary to resolve this issue.
The significance of the potential interaction of Rho family GTPases
with heterotrimeric G protein subunits remains unclear. Our results
are in agreement with a previous report that indicated that G
can
bind both RhoA and Rac1 and may be involved in membrane targeting of
these proteins (49). The putative Rho interaction surface at the
subunit N terminus has also been implicated in other protein
interactions such as binding of the Ste20 kinase and Cdc24 GEF in
S. cerevisiae (50, 51). It will be necessary to
examine the behavior of appropriate point mutants in suitable functional assays to assess the significance of these interactions.
Our results show that yeast Skn7 can interact with both yeast Rho1 and its mammalian homolog RhoA and that the ROCK-I/Kinectin homology region is required for this interaction. The ROCK-I/Kinectin homology is also required for the biological activity of Skn7 in vivo; in a number of assays for Skn7 function, deletion of this region inactivated the protein. SKN7 functions in the oxidative stress response (44) and in G1 cyclin synthesis (33), but its mechanism of action is not yet understood. Several observations suggest that it also plays a role in cell morphogenesis, possibly at the level of cell wall synthesis (31-33). In particular, skn7 pkc1 double mutants are inviable owing to massive lysis at the small-budded stage of the cell cycle (32, 33), a phenotype highly reminiscent of rho1 mutants (22). These observations suggest that Skn7, like Pkc1, might be a Rho1 effector, and consistent with this notion, high level overexpression of Skn7 is lethal owing to weakening of the cell wall (33), as would be expected if Skn7 were titrating Rho1. Our demonstration that Rho1 and Skn7 can physically interact and that the region of the protein that mediates the interaction is required for Skn7 function in vivo provides strong support for the idea that Skn7 is a Rho1 effector. Consistent with this, a multicopy plasmid expressing Rho1 partly suppresses the lethality induced by Skn7 overexpression.8
In summary, we have identified a new Diaphanous-related protein and the
putative exchange factor mNET1 as targets of RhoA and Cdc42. A region
of similarity between the RhoA-binding domains of ROCK-I and Kinectin
was used to identify related sequences in yeast Skn7 and the
heterotrimeric G protein subunit 2. These proteins were also shown
to interact with RhoA and Cdc42, and studies of Skn7 indicate that
these interactions are functionally significant in vivo.
Future studies will be directed toward the detailed characterization of
these interactions.
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ACKNOWLEDGEMENTS |
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We thank Peter Parker, Anne Ridley, Erik Sahai, and Shuh Narumiya for gifts of plasmids; Pablo Rodriguez-Viciana and Julian Downward for advice on GTPases; and John Sgouros for helpful discussions and data base analysis.
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FOOTNOTES |
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* This work was funded in part by the Imperial Cancer Research Fund and the Medical Research Council of Great Britain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by a postdoctoral fellowship from the Howard Hughes Medical Institute. To whom correspondence should be addressed. Present address: UCSF Cancer Center, Box 0128, San Francisco, CA 94143-0128. Tel.: 415-502-1713; Fax: 415-502-3179; E-mail: alberts{at}cc.ucsf.edu.
Supported in part by an International Travelling Research
Fellowship from the Wellcome Trust.
** Howard Hughes Medical Insitute International Research Scholar.
1
The abbreviations used are: MBS, myosin-binding
subunit; GST, glutathione S-transferase; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
GTPS, guanosine 5'-3-O-(thio)triphosphate; GEF, guanine
nucleotide exchange factor.
2 Flynn, P., Mellor, H., Palmer, R., Panayotou, G., and Parker, P. J. (1998) J. Biol. Chem. 273, 2698-2705.
3 A. S. Alberts and R. Treisman, unpublished data.
4 A. S. Alberts and R. Treisman, manuscript in preparation.
5 A. S. Alberts, unpublished data.
6 N. Bouquin and L. H. Johnston, unpublished data.
7 N. Bouquin and L. H. Johnston, manuscript in preparation.
8 N. Bouquin, unpublished data.
9 J. Sgouros, unpublished observations.
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REFERENCES |
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