(Received for publication, August 3, 1995)
From the
Rho, Rac, and Cdc42 are small GTPases that regulate the
formation of a variety of actin structures and the assembly of
associated integrin complexes, but little is known about the target
proteins that mediate their effects. Here we have used a motif-based
search method to identify putative effector proteins for Rac and Cdc42.
A search of the GenBank data base for similarity with the
minimum Cdc42/Rac interactive binding (CRIB) region of a potential
effector protein p65
has identified over 25 proteins
containing a similar motif from a range of different species. These
candidate Cdc42/Rac-binding proteins include family members of the
mixed lineage kinases (MLK), a novel tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and
several novel yeast and Caenorhabditis elegans proteins. Two
murine p65
isoforms and a candidate protein from C.
elegans, F09F7.5, interact strongly with the GTP form of both
Cdc42 and Rac, but not Rho in a filter binding assay. Three additional
candidate proteins, DPR2, MSE55, and MLK3 showed binding to the GTP
form of Cdc42 and weaker binding with Rac, and again no interaction
with Rho. These results indicate that proteins containing the CRIB
motif bind to Cdc42 and/or Rac in a GTP-dependent manner, and they may,
therefore, participate in downstream signaling.
Members of the Ras superfamily of small GTPases play a wide variety of cellular signaling roles that mediate proliferation and differentiation, cytoskeletal organization, protein transport, and secretion. The Ras GTPases have been studied most thoroughly, and now several components of the Ras signaling pathway have been identified using a combination of biochemical and genetic approaches(1, 2) . A related family of GTPases, the Rho subfamily, consists of three Rho genes, two Rac genes, Cdc42 and its close homologue G25K, rhoG, and TC10(3) . Early work in Saccharomyces cerevisiae, identified CDC42Sc as a protein required for bud emergence(4, 5) . In mammalian cells, the Rho subfamily members control the polymerization of actin and the assembly of focal complexes at the plasma membrane in response to extracellular signals(3, 6) . For example, microinjection of Rho into serum-starved Swiss 3T3 cells rapidly stimulates stress fiber and focal adhesion formation(7) , while Rac induces membrane ruffles (8) and Cdc42 induces the formation of filopodia(9) . In addition to their effects on the actin cytoskeleton, Rho GTPases also have a role in regulating kinase signaling pathways. For example, Rho, Rac, and Cdc42 stimulate a novel nuclear signaling pathway leading to transcriptional activation of the serum response element(10) . Rac and Cdc42, but not Rho, have also been shown to activate the c-Jun amino-terminal kinase (JNK) signaling pathway leading to c-Jun transcriptional activation (11, 12) . The mechanisms by which the Rho subfamily of GTPases regulate these apparently diverse biological processes is still not clear.
A large number of mammalian nucleotide exchange
factors (GEFs), related to the yeast exchange factor Cdc24 have been
identified including dbl, vav, ost, ect2, lbc, and Tiam1, and they may
provide tissue specificity or receptor-specific activation of Rho
family members(2, 3) . In addition, over 10 mammalian
GTPase-activating proteins (GAPs) ()for Rho family members
have been described including BCR, p50rhoGAP, chimaerin, ABR, p190-A,
p122, and myr-5(13) . The ability of these GAP proteins to
interact with GTPases in a GTP-dependent manner suggests that in
addition to being negative regulators, they may also act as effector
proteins. The multidomain nature of many of these GAPs further supports
this notion; for example, in addition to the GAP domain, p190 has a
GTPase domain(14) , p122 interacts with phospholipase
(15) , and the myosin family member, myr5, binds to
actin(16) .
The identification of effector proteins for
Rho-related GTPases is the first step toward defining their biological
activity, and a number of candidate proteins have already been
reported. The target protein involved in Rac-mediated activation of the
NADPH oxidase complex in phagocytic cells has been identified as
p67(17) . Although the Rac interactive site has
been mapped to the amino-terminal 199 amino acids of the protein, no
significant similarities have so far been found between this sequence
and other sequences in the data base. A tyrosine kinase containing a
SH3 domain, p120
, has been shown to bind specifically to
Cdc42 in a GTP-dependent manner, although its biological role is
unclear(18) . Another protein, the serine/threonine kinase
p65
, binds to both Cdc42 and Rac (but not Rho) in a
GTP-dependent manner, but again it is not known whether p65
mediates any of the described biological effects of these
GTPases(19, 20) . Interestingly, p120
and p65
do have some sequence similarities in a
region outside the kinase domain that represents an interactive site
for Cdc42/Rac GTPases(19) .
In this report, we have
localized the Cdc42 binding site of a murine p65 isoform
to a minimal conserved region of 16 amino acids. Using this small
protein motif, we have searched the GenBank
data bases and
identified 25 potential Cdc42- and/or Rac-binding proteins. In
vitro binding assays confirm that several of these proteins bind
to Cdc42 and/or Rac in a GTP-dependent fashion. Not all of these newly
identified proteins are kinases, suggesting a role for other types of
proteins in downstream signaling events mediated by the Rho family
proteins.
cDNA fragments were generated by restriction
enzymes or PCR and subcloned into the pGEX-4T-3 bacterial expression.
All constructs were confirmed by DNA sequence analysis. Fusion proteins
were made as glutathione S-transferase (GST) fusion proteins
induced in bacteria by
isopropyl-1-thio--D-galactopyranoside treatment, purified
on a glutathione-affinity column as described by the manufacturer
(Pharmacia Biotech Inc.), and checked for protein integrity by
SDS-polyacrylamide gel electrophoresis. The following constructs were
used: p65
-
(full-length, residues 1-545),
p65
-
-
1 (residues 29-546),
p65
-
-
2 (residues 118-546),
p65
-
-
3 (residues 29-90), DPR2 (residues
460-541), MSE55 (residues 11-120), F09F7.5 (residues
12-59), MLK3 (residues 454-538), and PLC-
1 (residues
560-726). Syn-1 was derived using two complementary
oligonucleotides containing the amino acid sequence EISLALREFHLNHVGLE
and subcloned in-frame into the EcoRI-XhoI site of
pGEX-4T-3.
Figure 1:
Delineation of a minimal Cdc42 binding
region in p65. GST fusion proteins were produced and
tested for p21 binding using the filter binding assay described under
``Materials and Methods.'' The location of the 16-amino acid
core CRIB sequence, also found in STE20 and p120
is
denoted by the black box.
Figure 2:
Sequence alignment of proteins containing
the CRIB motif. The accession number and/or name of each of the
proteins is shown on the left. The species origin is denoted
by: R, rat; M, mouse; H, human; Sc, S. cerevisiae; Sp, S. pombe; C, C.
elegans; B, bovine; D, Drosophila; and As, Ascaris. Amino acid sequence comparison is shown
between the different CRIB proteins. The number of residues matching
the strict consensus sequence of eight amino acid residues is also
shown. Sequence analysis suggests that C09B8.7 is a C. elegans homologue of p65. Binding of Cdc42 and Rac
determined experimentally is shown as positive (+) or
negative(-). p21 binding to p65
-
/R,
p65
-
/H, p120
, STE20, and Shk1 were
from Refs. 19, 20, 18, 19, and 38, respectively. Binding of Rac and
Cdc42 to WASP is from unpublished results (P. Aspenstrom, U. Lindberg,
and A. Hall, unpublished results). T23G5.3 is from (39) .
The CRIB motif occurs in both
kinases and non-kinase proteins. For example, one of the
CRIB-containing proteins, DPR2, is a Drosophila tyrosine
kinase(21) , which shows similarity in the kinase domain to
pp125, c-Abl, and p120
. Several
serine/threonine kinase including at least 3 isoforms of p65
and two human and three yeast kinases also contain a potential
CRIB motif. In addition, several members of the MLK family of
serine/threonine kinases also contain a protein sequence resembling a
CRIB motif. Two different yeast kinases encoded by X82499 and Z48149,
contain identical CRIB motifs. Non-kinase CRIB-containing proteins
include a human sequence MSE55 (and two related genes, accession
numbers T06431 and T75138/F12871), WASP, a human gene responsible for
Wiscott-Aldrich syndrome(28) , three different C. elegans genes (F09F7.5, T2365.3, and B0280.2) and two potential yeast
genes (P38785 and D9740.18).
Figure 3: Binding of Cdc42 and Rac to CRIB-containing proteins. 1 µg of various proteins were spotted onto nitrocellulose filters and incubated with 0.5 µg of radiolabeled Cdc42, Rac, or Rho. With the exception of MSP, all were GST fusion proteins. rhoGAP was used as a positive control in each case. Bound GTPase was visualized by autoradiography.
The
guanine nucleotide dependence of Cdc42 binding to these proteins was
investigated using a glutathione-agarose bead assay. Significant
binding of p65-
and -
, FO9F7.5, MSE55, DPR2,
MLK3, and Syn-1 was detected to the GTP-bound form of Cdc42 (Fig. 4), but not to the GDP-bound form of Cdc42. The results
are consistent with previous reports showing the GTP-dependent binding
of p120
(18) and p65
(19, 20) and are consistent with these proteins
being candidate effectors of Cdc42 and Rac biological activities.
Figure 4:
Proteins containing the CRIB motif bind to
Cdc42 in a GTP-dependent fashion. Approximately 5 µg (or 10 µg
of MLK3 and Syn-1) of each GST protein was immobilized to the
glutathione-Sepharose. V12 Cdc42 was loaded with either
[H]GDP or [
H]GTP by the
nucleotide exchange reaction and added to the proteins immobilized on
glutathione beads. Following washing, the resin was subjected to liquid
scintillation counting. The binding to each of the beads was expressed
as a percentage of input counts, typically around 2.5
10
cpm for both GDP- and GTP-bound
forms.
In
conclusion, we have delineated a short motif (the CRIB motif) in over
10 distinct proteins that confers binding to the Cdc42 and/or Rac
GTPases. At least another 15 proteins have been identified by a data
base search that contain a potential CRIB site. The length of the CRIB
motif is approximately 16 amino acids containing a region of variable
length between the two halves of the binding motif. The CRIB motif
contains eight core amino acids with the sequence
I-S-X-P-(X)-F-X-H-X-X-H-V-G.
It is interesting to note that proteins with one or two differences
within the core sequence can still show binding to Cdc42/Rac.
Of the
proteins shown to contain a functional CRIB motif, only the kinases
have a defined biochemical activity. In addition to the SH3-containing
kinase p120, a new Drosophila tyrosine kinase,
DPR2, was found to contain the CRIB motif. Although the CRIB domains
are found at a relatively similar distance after the kinase domains,
DPR2 is unlikely to be the Drosophila homologue of
p120
, since it lacks a SH3 domain and the overall amino
acid similarity is low. In addition to the p65
family of
kinases, a new serine/threonine kinase family, the MLK kinases, were
found to contain a potential CRIB motif. To date, there appears to be
at least four members of the MLK family including MLK1(30) ,
MLK2/MST(30, 31) ,
MLK3/SPRK/PTK1(32, 23, 33) , and
DLK(34) . All four of these kinases share a characteristic
hybrid kinase domain between serine/threonine and tyrosine kinases,
although SPRK/MLK3 (23) and DLK (34) have now been
shown to possess serine/threonine catalytic activity. In addition, all
four MLK members contain putative
-helical leucine zipper motifs
COOH-terminal to the kinase domain, although little is known of the
function of this region. One might expect that the activity of at least
MLK2 and MLK3 kinases will be stimulated following Cdc42/Rac binding as
seen with p65
(19, 20) , but this has yet
to be tested.
The biochemical function of the non-kinases containing the CRIB motif is not clear at all. Some of these proteins also contain proline-rich regions (e.g. F09F7.5 and MSE55) raising the possibility that they might act as adapters and interact with SH3-containing protein(s) or other polyproline-binding proteins such as profilin.
The identification of so many potential effectors for
Cdc42 and Rac is quite surprising. However, numerous activities have
already been ascribed to Rac and Cdc42, and each of these activities
may require a distinct effector(s). Rac regulates the assembly of focal
complexes and the polymerization of actin in lamellipodia, it has an
essential role in Ras-induced cellular transformation and can act as an
oncogene in its own right(35) , and it regulates the activity
of cPLA(36) . Rac and Cdc42 can also activate the
JNK kinase cascade (11, 12) and in Drosophila both Rac and Cdc42 have been shown to be involved in the extension
of neuronal growth cones(37) . The identification of proteins
containing the CRIB motif should aid in dissecting the molecular
mechanisms by which Cdc42/Rac GTPases regulate these processes.