(Received for publication, August 28, 1995; and in revised form, October 5, 1995)
From the
We previously reported the cloning of a serine/threonine kinase,
PAK (for p21 (Cdc42/Rac)-activated kinase), which binds to the
Ras-related GTPases Cdc42Hs and Rac1 (Manser, E., Leung, T.,
Salihuddin, H., Zhao, Z-s., and Lim, L.(1994) Nature 367,
40-46). These p21 proteins together with RhoA comprise the Rho
subfamily of proteins that are involved in morphological events. We now
report the isolation of a rat cDNA encoding a 150-kDa protein, which
specifically binds RhoA in its GTP form and contains an N-terminal
serine/threonine kinase domain highly related to the human myotonic
dystrophy kinase and a cysteine-rich domain toward the C terminus. The
RhoA binding domain is unrelated to other p21 binding domains. Antibody
raised against the kinase domain of the predicted protein, termed
ROK (for ROK
, RhoA-binding kinase), recognized a ubiquitous
150-kDa protein. The brain p150 purified by affinity chromatography
with RhoA exhibited serine/threonine kinase activity. In cultured
cells, immunoreactive p150 was recruited to membranes upon transfection
with dominant positive RhoA
mutant and was localized with
actin microfilaments at the cell periphery. These results are
consistent with a role for the kinase ROK
as an effector for RhoA.
The activation of cells by a number of growth factors through
their surface receptors specifically affect the cytoskeleton. Some of
these effects such as the formation of stress fibers(1, 2) lamellipodia(3) , and filopodia (4, 5) can be achieved by direct microinjection into
cells of specific p21 proteins of the Rho subfamily, indicating that
these p21 proteins act downstream of these growth factor receptors.
Very little information is available concerning the signaling pathways
beyond this point. A recent report has implicated a tyrosine kinase in
the RhoA-mediated formation of stress fibers(6) . In searching
for potential targets of different p21 proteins of the Rho family, we
developed methods for detecting direct interaction of the p21 proteins
with putative target proteins and isolated cDNAs encoding the tyrosine
kinase ACK ()(7) and the serine/threonine kinase
PAK(8) . ACK and PAK have related sequences responsible for the
interaction with Cdc42 and Cdc42/Rac, respectively(8) . We have
also demonstrated that there are cellular proteins that can
specifically interact with RhoA, with a p150 being ubiquitously present
in all tissues examined(8) . We now report the isolation and
characterization of a cDNA encoding this RhoA-binding protein. The
deduced amino acid sequence predicts a N-terminal serine/threonine
kinase domain homologous to myotonic dystrophy
kinase(9, 10) . A 90-amino acid region contains a
distinctive motif that binds RhoA in the GTP-bound form. Overexpression
of RhoA results in the recruitment of ROK
to peripheral membranes,
which is consistent with the kinase being a specific target for RhoA.
To determine the specificity and nucleotide dependence of p21
binding, GST/RhoA and GST/Cdc42 (8) were phosphorylated using
chicken protein kinase A (Sigma) and
[-
P]ATP (Amersham). The resulting labeled
p21 proteins (about 10
cpm/µg protein) were exchanged
with either GDP or GTP
S (Boehringer Mannheim) as described (7) for probing filters containing maltose-binding
protein/rbf-7 and maltose-binding protein/rbf-8 fusion proteins (0.1
µg/lane). The concentration of
P-labeled GST/RhoA and
GST/Cdc42 used in the binding assay was 0.1 µg/ml. After 30 min at
4 °C, filters were washed three times with ``wash
buffer'' (12) and exposed to Hyperfilm for 4-6 h.
Using GTP-Cdc42 as a probe for expression screening, we previously isolated a novel brain Cdc42-binding tyrosine kinase(7) . A similar approach was employed to identify cDNAs encoding proteins binding GTP-RhoA in a rat brain cDNA expression library. Six clones were obtained, which contained overlapping sequences, all of which encoded the RhoA binding domain (Fig. 1A). We then derived a small region (about 90 amino acids; rbf-8) responsible for the p21 binding (Fig. 1B). The sequence was dissimilar to the binding motifs for Cdc42/Rac1(8) . RhoA was only bound in its GTP form. Little or no Cdc42 was bound (Fig. 1C).
Figure 1:
Characterization of cDNAs encoding a
RhoA binding domain. A, six overlapping clones (rbf-1 to
rbf-6) encoding a RhoA binding domain, from a rat brain cDNA expression
library, were restriction-mapped and partially sequenced. Hc, HincII; Hd, HindIII; N, NdeI; P, PstI; Sp, SpeI; Sa, SalI; St, StuI; X, XbaI. Clones rbf-7 and rbf-8 were subclones of 3` HincII-HindIII and SpeI-HindIII
fragments from rbf-1, which also showed RhoA binding when expressed as
maltose-binding fusion proteins. B, the open reading frame of
rbf-8, encoding the RhoA binding domain. C, specificity of
RhoA binding to fusion proteins expressed from rbf-7 and rbf-8. RhoA or
Cdc42Hs expressed as GST/fusion proteins (8) were
phosphorylated with protein kinase A and
[-
P]ATP, exchanged with either GTP
S or
GDP, and used for binding to fusion proteins expressed from rbf-7 (upper band) and rbf-8 (lower
band).
The complete
sequence of a putative 159-kDa protein was obtained from analysis of
the overlapping cDNAs (Fig. 2A). It contained a novel
serine/threonine kinase most closely related to the human myotonic
dystrophy kinase (9, 10) (53% identity) and a p180
Cdc42-binding kinase (53% identity) recently identified in our
laboratory, ()as well as to the product of the fungal cot-1 gene (14) essential for hyphal elongation and
the Drosophila ``warts'' gene (15) implicated in cell growth and morphology (Fig. 2B). The RhoA-binding kinase (termed ROK
)
also contains a cysteine/histidine-rich domain at the C terminus (Fig. 2C); this domain had some similarity to those of
the PKC and chimaerin families(16) , but the spacing of the
invariant cysteines is not consistent with its being a diacylglycerol
receptor. Between the kinase and RhoA binding domains, the sequence is
predicted to assume an
-helical coil-coil structure (data not
shown).
Figure 2:
Sequence of a novel kinase containing a
RhoA binding domain and other domains. A, predicted amino acid
sequence of rat brain RhoA-binding protein. Sequences were obtained
from both directions of two overlapping clones as well other cDNAs
isolated by expression screening (rbf-1 to rbf-6). Restriction sites
refer to those shown in Fig. 1. The N-terminal kinase, p21
binding (BD) and cysteine-rich (CR) domains are shown
in bold. A diagrammatic representation of the predicted
protein is also depicted. B, alignment of ROK kinase
domain and related kinases. ROK
was aligned with human myotonic
dystrophic kinase (DMK, GenBank(TM) accession no. L08835), Neurospora cot-1 (N.Cot-1, accession no. P38679), Drosophila warts gene product (wts, accession no.
L39837), and rat PKC
(accession no. P28867) using the CLUSTAL
method (DNASTAR). C, alignment of cysteine/histidine-rich
domains (CRD) of ROK
, rat PKC
, human Raf-1, and rat n-chimaerin. The invariant residues present in most CRDs of
this class (16) are in bold and marked with asterisks. The position of His as a potential substitute for
consensus Cys is marked (+).
We have been unable to express full-length recombinant
ROK protein in Escherichia coli. However, the kinase
domain could be expressed as a fusion protein with maltose-binding
protein which was used to raise polyclonal antiserum. An immunoreactive
150-kDa protein was detected in all tissues, including brain as well as
cultured rodent and human cells (Fig. 3A), which
appeared to correspond to a ubiquitous p150 RhoA-binding protein
previously described(8) . The native 150-kDa RhoA-binding
protein was then purified from rat brain cytosol by affinity
chromatography with GST/RhoA fusion protein. The purified protein bound
RhoA and was recognized by the antiserum to ROK
(Fig. 3A, lane E3). The p150 was capable of
autophosphorylation and of phosphorylating myelin basic protein at
serine and threonine residues (Fig. 3B). Although
GTP-RhoA did not appear to stimulate p150 kinase activities in
vitro, it is possible that the p150 kinase (ROK
) becomes
activated by affinity chromatography with RhoA during the purification
and thus incapable of responding further to RhoA.
Figure 3:
Expression of ROK in various tissues,
its purification from brain, and its kinase activities. A,
RhoA binding and ROK
immunoreactivity of soluble extracts and
purified ROK
. Soluble protein (150 µg) from rat brain (BR) and cultured cells (NB, human neuroblastoma; C6, rat C6 glioma; Gr, rat cerebellar granule cells; FB, mouse NIH3T3 fibroblasts) separated on a 9% polyacrylamide
gel were probed with [
-
P]GTP-GST/RhoA (upper panel) or polyclonal antibodies raised against ROK
kinase domain (lower panel). The last lane is Coomassie
staining of purified rat brain p150 isolated by RhoA-affinity
chromatography (see ``Materials and Methods''), which
exhibits ROK
immunoreactivity and RhoA binding (lane E3). B, kinase activity of p150 ROK
. Kinase assays were
carried out in the absence (lanes 1 and 3) and
presence of GTP
S-RhoA (lanes 2 and 4). Assays in lanes 3 and 4 were performed with myelin basic
protein (MBP) as exogenous substrate. For phosphoamino acid
analysis,
P-phosphorylated p150 (lane 1) and
myelin basic protein (lane 2) were subjected to analysis as
described previously(8) , using phosphoserine (PS),
phosphothreonine (PT), and phosphotyrosine (PY) as
standards.
We then examined
the effect of overexpressing RhoA on the intracellular distribution of
the p150 ROK in Cos-7 cells. About 20% of the cells were
efficiently transfected. ROK
immunoreactivity, detected with a
specific monoclonal antibody, was mainly present in the cytosolic
fraction in control cells transfected with empty vector (Fig. 4A). Upon transfection with either RhoA or
RhoA
, both of which are effective in promoting stress
fiber formation when microinjected into cells(1) , there was an
increase in p150 ROK
in the pellet fractions, suggesting that RhoA
promoted its association with membranes. This association was
investigated at the cytological level using HeLa cells transfected with
the dominant-positive RhoA
mutant. In HeLa and other
cells examined, ROK
was distributed in the cytoplasm.
RhoA
-transfected cells showed a generally rounded
morphology with increased actin microfilaments mainly at the cell
periphery (Fig. 4B, panel b), but also present
in stress fibers adjacent to the substratum (Fig. 4B, panel c). Much of the endogenous ROK
immunoreactivity
co-localized with the actin microfilaments at the peripheral cell
membrane (Fig. 4B, panel a), but not with
stress fibers (data not shown). By contrast, in cells expressing
Cdc42
no such membrane translocation occurred with
ROK
immunoreactivity being generally cytosolic (Fig. 4B, panel f). Neither was PKC
(analyzed as another control) membrane-associated in
RhoA
- transfected cells (data not shown). We conclude
that there is a specific recruitment of ROK
to plasma membrane
with activated RhoA.
Figure 4:
RhoA-dependent membrane association of
ROK. A, Cos-7 cells were transiently transfected with
vector containing a HA tag alone (panel 1), or vector with
either RhoA (panel 2) or RhoA
(panel
3). Soluble (s) and pellet (p) fractions (100
µg) from transfected cells were separated on 12% SDS-polyacrylamide
gels, transferred to nitrocellulose for Western blotting using
antibodies to ROK
or anti-HA for the p21 proteins. B, in panels a-c, HeLa cells were transiently transfected with
the vector containing HA-tagged RhoA
. After 16 h, cells
were fixed and stained with monoclonal antibody 1A1 for ROK
(a). Actin microfilament distribution at the same (confocal)
level of the membrane where ROK
was localized upon RhoA
expression is shown in b. Stress fibers of these cells
are located at their base in contact with the substratum (c).
Cells expressing RhoA
(d and e) or
Cdc42
(f and g) were double-stained for
ROK
(d and f) and for the HA-tagged p21 proteins (e and g). Low levels of p21 expression were
sometimes not detectable with HA staining. Bar = 10
µm.
ROK is a member of a serine/threonine kinase family,
which includes the myotonic dystrophy kinase (9, 10) Neurospora cot-1, and the Drosophila warts gene(15) , as well as a recently
isolated Cdc42-binding kinase.
The myotonic dystrophy
kinase may be involved in membrane functions related to ion channels (17, 18) while cot-1 and warts mutants show abnormal cell growth with morphological consequences.
It is intriguing that the related mammalian ROK
contains a RhoA
binding domain. This domain is unrelated to the other known p21 binding
domains and does not affect intrinsic or p190 RhoGAP-stimulated GTPase
activities of RhoA (data not shown), unlike PAK or ACK, which inhibit
the GTPase activities of their p21 partners(7, 8) .
RhoA, which shares 50% identity with Cdc42 and Rac1, binds to a
different protein population than the latter p21 proteins, with p150
being ubiquitous and most prominent in all tissues (8) . This
p150 RhoA-binding kinase (putatively identified as ROK
) is readily
purified using a GST-RhoA affinity column.
In relation to the in
vivo effects of RhoA in promoting membrane association of
ROK, we have found that HA tagged-ROK
is activated when
co-transfected with RhoA on enzymatic analysis of HA-immunoprecipitates
(data not shown). Most cells with membrane-associated ROK
showed a
more rounded morphology, with the kinase being co-localized with
peripheral actin microfilaments. No correlation of ROK
localization with stress fibers was observed, although the latter are
increased in RhoA-overexpressing cells. This suggests that ROK
may
not be directly involved in regulating or maintaining stress fibers.
However, apart from increased stress fibers a frequent consequence of
RhoA transfection is the occurrence of cells with rounded morphology.
Filamentous actin reorganization is essential in the rounding up
process and in the Rho-dependent formation of the contractile ring,
which precedes cell division(19) , and it is possible that
ROK
participates in these events. In addition to morphology and
cytokinesis(19, 20) , evidence is accumulating for the
involvement of Rho proteins in motility(21) ,
transformation(22, 23) , and apoptosis(24) .
Our finding of a RhoA-binding kinase should prove helpful in
determining the mechanisms underlying these important cellular
activities as well as the pathological basis of myotonic dystrophy
because of the similarity of the dystrophic kinase to ROK
.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U38481[GenBank].