(Received for publication, June 27, 1996, and in revised form, October 17, 1996)
From the PKN is a fatty acid- and Rho-activated
serine/threonine protein kinase, having a catalytic domain homologous
to protein kinase C family. To identify components of the PKN-signaling
pathway such as substrates and regulatory proteins of PKN, the yeast
two-hybrid strategy was employed. Using the N-terminal region of PKN as
a bait, cDNAs encoding actin cross-linking protein PKN is a serine/threonine protein kinase, having a catalytic
domain homologous to protein kinase C family in the C terminus and a
unique regulatory region in the N terminus (1, 2). The N-terminal
region of PKN contains repeats of leucine zipper-like motif, suggesting
promotion of protein-protein association through hydrophobic
interactions (3). We demonstrated that Rho, a small GTP-binding
protein, binds to PKN in a GTP-dependent fashion and that
this binding leads to the activation of PKN, suggesting that PKN is one
of the targets of Rho (4, 5). In order to identify other proteins that
interact with PKN, we have used a yeast two-hybrid system with the
N-terminal region of PKN as bait. One of the positive cDNA clones
isolated from human brain cDNA library encoded a neurofilament L
protein, a neuron-specific intermediate filament protein (6). We have
demonstrated that PKN binds to and phosphorylates the head-rod domain
of intermediate filament proteins such as each subunit of neurofilament
and vimentin in vitro (6) and raised the possibility that
PKN plays a role in the assembly of intermediate filament, one of the
major components of cytoskeleton. Here we report that the two other
groups of positive cDNA clones encoded Schemes of the fusion constructs for human PKN, human
skeletal muscle type Schematic representation of the various
expression constructs and results of their interactions in the
two-hybrid system. The schematic whole structure of each protein
is represented at the top of each figure, and the deletion
mutants of each protein are aligned below. The
numbers preceding and following each line denote the
positions of the most terminal aa residue of each clone, which is
represented by solid or open box. The interaction
in the two-hybrid system was examined by a filter assay for
cDNAs encoding the
spectrin-like repeat 3 (aa 479-600) and EF-hand-like region (aa
712-834) of HuActNm were amplified by PCR from the human brain
cDNA library and were ligated to pGEX4T vector. cDNAs encoding
the spectrin repeat 20 and EF-hand-like region of A plasmid for
in vitro transcription of the full-length coding region of
HuActSk1 was constructed as follows. The cDNA encoding the
N-terminal region (aa 1-422) of HuActSk1 containing actin-binding domain was amplified by PCR from the human brain cDNA library and
was ligated to the C-terminal part (aa 423-894) of clone 4 isolated in
the two-hybrid screening. This cDNA for the full-length coding
region of HuActSk1 was subcloned into pBluescript II SK+. The plasmid
was linearized by cutting with XhoI, and the cRNA was
transcribed using T3 RNA polymerase. In vitro transcription for the N-terminal region of PKN (aa 1-474, this region is designated as PKNN2 (6)) was performed as described previously (6). For in
vitro translation, cRNAs were translated in rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine as
described previously (6).
-For the in vitro binding
experiment, 2 µl of in vitro translated PKNN2 was mixed
with 5 µg of each GST- For analysis of the effect of phosphatidylinositol 4,5-bisphosphate
(PI4,5P2) (Boehringer Mannheim) on the binding between The anti-hemagglutinin (HA) monoclonal antibody
12CA5 was purchased from Boehringer Mannheim. HA-tagged cDNA for HuActSk1 (aa
333-894) was created by fusion of a cDNA encoding 9-aa epitope
from the influenza HA to the N terminus of clone 4. A vector pHA-Act
was constructed by subcloning this cDNA into pTB701 (8). Empty pHA
vector was constructed by subcloning a cDNA encoding only HA
epitope into pTB701. A vector pHA-Act or empty pHA vector was
cotransfected into COS7 cells with the expression vector pMhPKN3 (2)
encoding the full-length human PKN. After 48 h, ~106
cells were lysed in 500 µl of lysis buffer (20 mM
Tris/HCl at pH 7.5, 1% Nonidet P-40, 137 mM NaCl, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml
aprotinin, 10 µg/ml leupeptin) for 1 h. Insoluble materials were
removed by centrifugation at 15,000 × g for 10 min.
Twenty-five-µl aliquot of the supernatant was subjected to SDS-PAGE,
and the amount of the expression of proteins was assessed by Western
blotting using 12CA5 and Actin was
purified from rabbit skeletal muscle by the method of Mommaerts (10).
The phosphorylation by PKN was carried out at
30 °C in an assay mixture containing 20 mM Tris/HCl at
pH 7.5, 4 mM MgCl2, 100 µM ATP,
185 kBq of [ We screened a million yeast colonies transformed with both
human brain cDNA library fused to Gal4 transcriptional activation domain and a bait construct encoding PKNN1 fused to Gal4 DNA binding domain. The 82 plasmids were isolated representing 16 different cDNAs as judged by cDNA sequencing. Three positive clones
(clones 4, 10, and 25) encoded the skeletal muscle type
A number of distinct isoforms of
The interaction of
We
investigated whether the binding of Since PKN bound to
The actin cytoskeleton plays a critical role in a number of
cellular processes including motility, chemotaxis, and cell division (32-35). Members of Rho family of small GTP-binding proteins have been
implicated in the regulation. Rho promotes the formation of actin
stress fibers and focal adhesions (36, 37), although the mechanism by
which Rho mediates the effect on the actin cytoskeleton is unclear.
Recently it has shown that Rho interacts physically and regulates the
activity of PI(4)P5-kinase in mouse fibroblasts and thereby regulates
the cellular levels of PI4,5P2 (38, 39). PI4,5P2 can regulate in
vitro the interactions of a number of actin-binding proteins, such
as We thank Dr. Y. Nishizuka for
encouragement.
Radioisotope Research Center,
-actinin,
which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and
-actinin was determined by in vitro
binding analysis using the various truncated mutants of these proteins.
The N-terminal region of PKN outside the RhoA-binding domain was
sufficiently shown to associate with
-actinin. PKN bound to the
third spectrin-like repeats of both skeletal and non-skeletal muscle
type
-actinin. PKN also bound to the region containing EF-hand-like
motifs of non-skeletal muscle type
-actinin in a
Ca2+-sensitive manner and bound to that of skeletal muscle
type
-actinin in a Ca2+-insensitive manner.
-Actinin
was co-immunoprecipitated with PKN from the lysate of COS7 cells
transfected with both expression constructs for PKN and
-actinin
lacking the actin-binding domain. In vitro translated
full-length
-actinin containing the actin-binding site hardly bound
to PKN, but the addition of phosphatidylinositol 4,5-bisphosphate,
which is implicated in actin reorganization, stimulated the binding
activity of the full-length
-actinin with PKN. We therefore propose
that PKN is linked to the cytoskeletal network via a direct association
between PKN and
-actinin.
-actinin, a constituent
of the other major component of cytoskeleton.
Two-hybrid Screens and Constructs for Two-hybrid
Systems
-actinin
(HuActSk1,1 designated in Ref. 7), and
non-skeletal muscle type
-actinin (HuActNm, designated in Ref. 7)
used in this study were represented in Fig. 1. Screening of the
associated proteins with the N-terminal region of human PKN (aa 1-540,
this region is designated as "PKNN1") was performed as described
previously (6). Primary positive clones were recovered and
retransfected into the original yeast host strain YGH1
(a, ura3-52, his3-200,
ade2-101, lys2-801, trp1-901,
leu2-3, Canr, gal4-542,
gal80-538,
LYS2::gal1uas-gal1tata-HIS3,
URA3::gal1-lacZ) in combination with
the Gal4bd-PKN or the Gal4bd-p53 tumor suppressor. Library plasmids
that activated marker expression only in the presence of PKN were
analyzed further.
Fig. 1.
-galactosidase activity. "+++" and "+" indicate the
development of blue color within 20 min and 24 h from initiation
of the assay, respectively. "±" indicates the development of faint
blue color after 24 h from initiation of assay, and "
"
indicates no development of color within 24 h. Gal4bd and LexAbd
indicate the DNA binding domain of Gal4 and LexA, respectively.
Gal4ad and VP16ad indicate the transcription
activation domain of Gal4 and VP16, respectively. A, human
PKN. HuActSk1 (skeletal muscle type
-actinin) (aa 423-894) was
expressed as a fusion protein with VP16 transcription activation domain, and its interaction with the various deletion mutants of PKN
expressed as fusion proteins with the LexA DNA binding domain was
examined in the two-hybrid system. LZ indicates the leucine
zipper-like motif. BR indicates the region rich in basic aa.
Solid box indicates the bait construct. B,
HuActSk1. The numbers (4, 10, and 25)
on the right indicate the clone numbers isolated in the
screening. The N-terminal region of PKN (aa 1-540; this region was
designated as PKNN1) was expressed as a fusion protein with Gal4 DNA
binding domain, and its interaction with the various deletion mutants
of HuActSk1 expressed as a fusion protein with Gal4 activation domain
or VP16 activation domain was examined in the two-hybrid system.
SR indicates spectrin-like repeats. C, HuActNm
(non-skeletal muscle type
-actinin). SR indicates spectrin-like repeats.
[View Larger Version of this Image (23K GIF file)]
-spectrin were
amplified by PCR from rat brain cDNA library and were ligated to
pGEX4T vector. Expression and purification of GST or GST fusion
proteins were performed according to the manufacturer's instruction
(Pharmacia Biotech Inc.) The eluate from glutathione-Sepharose 4B
(Pharmacia Biotech Inc.) was dialyzed overnight against 10 mM Tris/HCl at pH 7.5 containing 1 mM EDTA, 1 mM DTT, and 0.1 µg/ml leupeptin.
-actinin fusion protein or with 25 µg of
GST alone in 400 µl of GST binding buffer (20 mM Tris/HCl
at pH 7.5, 0.5 mM DTT, 150 mM NaCl, 0.05%
Triton X-100, 1 mM EDTA, 1 µg/ml leupeptin) and incubated
for 1 h at 4 °C. After addition of 25 µl of
glutathione-Sepharose 4B pretreated with 10 mg/ml Escherichia
coli extract to block nonspecific binding, the binding reaction
was continued for an additional 30 min at 4 °C. The
glutathione-Sepharose 4B was then washed four times in GST wash buffer
(20 mM Tris/HCl at pH 7.5, 0.5 mM DTT, 1 mM EDTA, 1 µg/ml leupeptin) containing 0.5 M
NaCl and 0.5% Triton X-100 and further washed with GST wash buffer.
Bound proteins were eluted with GST elution buffer (100 mM
Tris/HCl at pH 7.5, 10 mM glutathione, 120 mM
NaCl, 1 mM EDTA, 0.5 mM DTT, 1 µg/ml leupeptin) and were subjected to SDS-PAGE.
-actinin and
PKN, 2 µl of in vitro translated full-length HuActSk1 was
mixed with 5 µg of GST-PKNN1 fusion protein or with 25 µg of GST
alone in 400 µl of buffer P (20 mM Tris/HCl at pH 7.5, 0.5 mM DTT, 120 mM NaCl, 1 mM EDTA)
and incubated for 1 h at room temperature with or without PI4,5P2
as indicated in the figure legends. After addition of 25 µl of
glutathione-Sepharose 4B pretreated with E. coli extract,
the binding reaction was continued for an additional 30 min at 4 °C.
The glutathione-Sepharose 4B was then washed four times in buffer P
containing 0.01% Triton X-100. Bound proteins were eluted with GST
elution buffer and were subjected to SDS-PAGE. The binding was
visualized and quantitated by an imaging analyzer (FUJI BAS1000).
C6, a specific
antiserum against PKN, was prepared by immunizing rabbits with the
bacterially synthesized fragment of aa 863-946 of rat PKN.
C6. Four hundred-µl aliquot of the
supernatants was incubated with 1 µg of 12CA5 for 2 h. After
addition of 20 µl of 50% protein A-Sepharose, the mixtures were
further incubated for 1 h. The immunoprecipitates adsorbed to
protein A-Sepharose were washed twice with HA wash buffer (100 mM Tris/HCl at pH 7.5, 0.5 M LiCl) and twice
with 10 mM Tris/HCl at pH 7.5. The resultant
immunoprecipitates were resuspended in 50 µl of Laemmli's sample
buffer (9), and 25-µl aliquot of the extract was subjected to
SDS-PAGE, following detection by immunoblotting with
C6.
-Actinin was purified from bovine aorta by the method of Feramisco
and Burridge (11). Vinculin was purified from bovine aorta by the
method of Kobayashi and Tashima (12). Caldesmon was partially purified
from bovine aorta by the method of Abe et al. (13). Filamin,
metavinculin, and talin were partially purified from bovine aorta as
described (11).
-32P]ATP, phosphate acceptors, 20 ng/ml
purified PKN from rat testis (13), and with or without 40 µM arachidonic acid as indicated in each experiment.
Partial purified protein was boiled for 3 min to destroy endogenous
kinase activity before use as a phosphate acceptor. After incubation
for 5 min, the reaction was terminated by the addition of an equal
volume of Laemmli's sample buffer and separated on SDS-polyacrylamide
gels. The gels were dried under vacuum, and the phosphorylation was
visualized and quantitated by an imaging analyzer (FUJI BAS1000). When
the
protein kinase C peptide (1) was used as a phosphate acceptor,
reactions were terminated by spotting a mixture onto a Whatman P81
paper and submerging it in 75 mM phosphate and followed by
three 10-min washes. Incorporation of [32P]phosphate into
the peptide was assessed by scintillation counting.
PKN Interacts with -Actinin in the Yeast Two-hybrid
System
-actinin
(HuActSk1, designated in Ref. 7). The clone 4 encoded HuActSk1 from aa 333 to the C terminus, and both clone 10 and clone 25 encoded HuActSk1
from aa 344 to the C terminus. All these clones contained complete C
terminus but lacked the N-terminal actin-binding domain (14) (Fig.
1B). These clones resulted in high
-galactosidase levels upon co-transformation with the PKN bait
construct in the original yeast host strain YGH1. The specificity of
this interaction was tested further by measuring the stability of other
combinations of two-hybrid constructs, LexAbd (instead of Gal4bd)-PKN
and Gal4ad-
-actinin to support lacZ expression in L40
cells (MATa trp1 leu2 his3 LYS2::lexA-HIS3
URA3::lexA-lacZ). As shown in Fig. 2,
high
-galactosidase activity was also developed in this system,
suggesting a specific interaction between the N-terminal region of PKN
and
-actinin. The two-hybrid method was employed to identify the region on PKN that interacted with HuActSk1, and this region was compared with the binding site for RhoA, protein already known to
interact with PKN in vitro and in vivo (4, 5).
The RhoA-binding site has been mapped on the aa 33-111 of PKN that
corresponds to the first leucine zipper-like sequence of PKN (15),
whereas
-actinin very weakly interacted with this region of PKN
(Fig. 1A). By contrast,
-actinin strongly interacted with
aa 136-189 of PKN, whereas no interaction was detected between RhoA
and this region of PKN (data not shown). This region corresponds to the second leucine zipper-like sequence and its immediate N-terminal region, which is conserved through evolution in vertebrates (16). Thus
-actinin binds most avidly to the region distinct from that which
binds to RhoA. These results raise the possibility that PKN binds
simultaneously to RhoA and
-actinin.
Fig. 2.
Interaction between PKN and HuActSk1 in the
two-hybrid system. PKNN1 (lanes 1-4) or murine tumor
suppressor p53 (aa 72-390) (lanes 5-8) was expressed as a
fusion protein with LexA DNA binding domain, and its interaction with
SV40 large T antigen (aa 84-708) (lanes 1 and
5), clone 21 protein (lane 2 and 6), clone 4 protein (lane 3 and 7), and clone 10 protein (lane 4 and 8) expressed as fusion
proteins with the Gal4 activation domain was examined by a filter assay
for -galactosidase activity. The clone 21 encodes the head-rod
domain of neurofilament L protein (6). Shown are developments of blue
color 1 h after initiation of the filter assay of independent
colonies picked from selected plates lacking Trp and Leu.
[View Larger Version of this Image (67K GIF file)]
-Actinin is composed
of three domains, an N-terminal actin-binding domain, extended
rod-shaped domain with four internal 122 aa repeats (spectrin-like
repeats), and a C-terminal region containing a pair of presumptive
helix-loop-helix Ca2+-binding motifs, often referred to as
EF-hands (reviewed in Ref. 17). To investigate whether PKN binds
directly to
-actinin and to clarify which part of
-actinin is
necessary for binding to PKN, various truncated constructs of HuActSk1
were produced as GST fusion proteins in E. coli (Fig.
1B). As shown in Fig. 3, in vitro
translated PKNN2 strongly bound to each
-actinin fragment (aa
423-653, aa 653-837, and aa 486-607) but not to the fragment (aa
837-894 and aa 604-719). The ability of the binding was verified by
the demonstration that the complex was resistant to washing with 0.5%
Triton X-100, 0.5 M NaCl. These results suggest that the
recombinant
-actinin interacts directly with recombinant PKN and
that two distinct regions of HuActSk1, which correspond to the
spectrin-like repeat 3 and the region containing the EF-hand-like motifs, play important roles in the recognition of PKN. As expected from the two-hybrid data, the N-terminal region of PKN (aa 136-474) translated in vitro, which lacked the RhoA binding region,
was sufficient for direct binding to
-actinin (Fig. 3, lanes
18-20).
Fig. 3.
In vitro binding analysis of
interaction between PKN and HuActSk1. 35S-Labeled
in vitro translated N-terminal region of PKN (aa 1-474, this region was designated as PKNN2; lanes 1-7 and
11-17) or PKNBal (aa 136-474; lanes 8-10
and 18-20) was incubated with the bacterially synthesized
GST or GST-fused various deletion mutants of HuActSk1 as indicated in
Fig. 1B. Aliquots of the initial binding reaction mixtures
(10 µl) were removed before precipitation and applied to
electrophoresis, which were indicated as Input shown in
the upper panel. GST or GST-fused proteins were collected
with glutathione-Sepharose beads, analyzed by 10% SDS-PAGE, and
followed by autoradiography, which were indicated as
+G-beads shown in the lower panel. Lanes 1 and
11, GST-HuActSk1(333-423); lanes 2, 8, 12, and
18, GST-HuActSk1(423-653); lanes 3, 9, 13, and
19, GST-HuActSk1(653-837); lanes 4 and
14, GST-HuActSk1(837-894); lanes 5 and
15, GST-HuActSk1(486-607); lanes 6 and
16, GST-HuActSk1(604-719); lanes 7, 10, 17, and
20, GST. White arrowhead indicates the position
of labeled PKNN2, and black arrowhead indicates the position
of the labeled PKN
Bal. Molecular mass markers are indicated in
kDa.
[View Larger Version of this Image (42K GIF file)]
-Actinin
(HuActNm) in Vitro and Ca2+ Dependence of Its
Interaction
-actinin have
been characterized, including skeletal, smooth, and non-muscle
-actinins, from various kinds of cells and tissues. The only
recorded functional difference among these
-actinins is that binding
of the non-muscle isoform to F-actin is inhibited by Ca2+,
whereas binding of the muscle isoform is Ca2+-insensitive
(18-21). In human, only one clone of the non-muscle cytoskeletal
isoform (HuActNm, designated in Ref. 7), having strong sequence
homology with HuActSk1 (89% similarity and 80% identity for pairwise
comparison), has been isolated (22, 23). Then we tested whether PKN
could bind to the region of HuActNm corresponding to the PKN-binding
site of HuActSk1. As shown in Fig. 4A, PKN
could also bind to spectrin-like repeat 3 domain of HuActNm, whereas
the binding to the EF-hand-like domain of HuActNm was not detected in
the absence of Ca2+. However, PKN could effectively bind to
the EF-hand-like region of HuActNm in the presence of 1 mM Ca2+ (Fig. 4B). Although it is
uncertain at present whether this Ca2+ dependence is
retained in the binding between PKN and the full-length HuActNm, the
Ca2+ dependence may be one of the reasons why the cDNA
clone encoding the non-muscle type
-actinin was not isolated in the
two-hybrid screening of "brain" cDNA library. Beggs et
al. (7) compared the sequences of EF-hand-like regions of
HuActSk1 with the EF-hand consensus of Kretsinger (24) and
indicated that the first EF-hand-like region of HuActSk1 has only 11/16
matches with either an arginine or lysine at the Y position
and that these peptides would probably not be able to coordinate
Ca2+ binding properly. Our results support this estimation
from a different point of view.
Fig. 4.
Interaction of PKN with HuActNm.
35S-Labeled in vitro translated PKNN2 was
incubated with the bacterially synthesized GST or GST-fused various
deletion mutants of HuActNm as indicated in Fig. 1C.
Aliquots of the initial binding reaction mixtures (10 µl) were
removed before precipitation and applied to electrophoresis, which were
indicated as Input. GST or GST-fused proteins were collected
with glutathione-Sepharose beads, analyzed by 10% SDS-PAGE, and
followed by autoradiography. White arrowhead indicates the position of labeled protein. Molecular mass markers are indicated in
kDa. A, specific interaction of PKN with the deletion
mutants of HuActNm. Lanes 1 and 4, GST-HuActNm
(aa 479-600); lanes 2 and 5, GST-HuActNm (aa
712-834); lanes 3 and 6, GST. B,
effect of Ca2+ on the interaction of EF-hand-like region of
HuActNm. GST-HuActNm (aa 712-834) was incubated with in
vitro translated PKN in the absence of Ca2+
(lanes 1 and 3) or in the presence of 1 mM Ca2+ (lanes 2 and
4).
[View Larger Version of this Image (19K GIF file)]
-Actinin
-Actinin is a member of spectrin superfamily,
including spectrin, dystrophin, and so on (17, 25, 26). Family members are characterized by the N-terminal actin-binding domain, central rod-shaped spectrin-like repeats, and the C-terminal EF-hand-like domain.
-Spectrin contains 21 rod-shaped repeats in the N-terminal to the EF-hand-like domain. The C terminus of
-spectrin is clearly related to
-actinin, and especially the repeat 20 of
-spectrin has extensive homology to the repeat 3 of
-actinin (27, 28), and the
position of the repeat in each protein seems to be related to each
other. Since PKN bound to the repeat 3 of
-actinin, we examined
whether PKN can bind to the repeat 20 of
-spectrin. As shown in Fig.
5, in vitro binding between PKN and the
repeat 20 of rat
-spectrin was not detected in the same condition in which PKN bound to the repeat 3 of
-actinin. These results indicate that PKN specifically binds to the spectrin-like repeat of
-actinin.
Fig. 5.
Specific interaction of PKN with
spectrin-like repeats of -actinin. 35S-Labeled
in vitro translated PKNN2 was incubated with the bacterially synthesized GST or GST-fused spectrin-like repeats of
-actinin or
spectrin repeat of
-spectrin. Aliquots of the initial binding reaction mixtures (10 µl) were removed before precipitation and applied to electrophoresis, which were indicated as Input.
GST or GST-fused proteins were collected with glutathione-Sepharose beads, analyzed by 10% SDS-PAGE, and followed by autoradiography. White arrowhead indicates the position of the labeled
protein. Molecular mass markers are indicated in kDa. Lanes
1 and 5, GST-spectrin-like repeat 3 of HuActSk1 (aa
486-607); lanes 2 and 6, GST-spectrin-like repeat 3 of HuActNm (aa 479-600); lanes 3 and 7,
GST-spectrin repeat 20 of
-spectrin; lanes 4 and
8, GST.
[View Larger Version of this Image (42K GIF file)]
-Actinin in Vivo
-actinin with PKN in vivo was examined by cotransfection
experiment in COS7 cells (Fig. 6). An epitope-tagged
-actinin was generated by fusion of a 9-aa epitope from the
influenza HA to the N terminus of clone 4 protein, enabling the
selective immunoprecipitation of the tagged
-actinin polypeptide
with anti-HA monoclonal antibody 12CA5 (29). This HA-tagged
-actinin
contains the complete C-terminal region of
-actinin, whereas it
lacks the N-terminal actin-binding domain. After co-expression of
HA-tagged
-actinin with the full-coding region of PKN in COS7 cells,
anti-HA immunoprecipitates contained substantially immunoreactive PKN. These results suggest that the C-terminal region of
-actinin can
associate in vivo with PKN.
Fig. 6.
In vivo interaction of PKN with
HuActSk1. A vector pHA-Act encoding HA epitope fused to the aa
333-894 of HuActSk1 (lanes 1, 3 and 5) or empty
pHA vector (lanes 2, 4, and 6) was cotransfected
into COS7 cells with the expression vector pMhPKN3 (2) encoding the
full-length human PKN. Cells were extracted, and recombinant
polypeptides were immunoprecipitated using anti-HA antibody 12CA5. PKN
and HuActSk1 in each extract (lanes 3-6) and in each
immunoprecipitates (lanes 1-2) were subjected to SDS-PAGE and detected by immunoblotting with anti-PKN antiserum C6
(lanes 1-4) and 12CA5 (lanes 5 and
6), respectively. White arrow indicates the
position of PKN. Black arrow indicates the position of
HA-HuActSk1. Molecular mass markers are indicated in kDa.
[View Larger Version of this Image (35K GIF file)]
-Actinin
-Actinin in vivo bound to various
amounts of endogenous PI4,5P2, and the specific interaction between
-actinin and PI4,5P2 regulates the F-actin-gelating activity of
-actinin (30). This indicates that PI4,5P2 causes a conformational
change in
-actinin. Exogenously added PI4,5P2 can bind to
-actinin strongly, and the binding is tight and stable (30). Then we
examined the binding activity of PKN with
-actinin in the presence
or absence of PI4,5P2. Since PI4,5P2 binding region resides in the
actin-binding domain of
-actinin (14), in vitro
translated full-length
-actinin containing actin-binding domain was
used in this in vitro binding experiment (Fig.
1B). Interestingly, the full-length
-actinin very weakly
but specifically bound to PKN in the absence of PI4,5P2. However,
addition of 10 µM PI4,5P2 stimulated the binding of the full-length
-actinin to PKN (Fig. 7A).
Therefore PI4,5P2 appears to influence the conformation of
-actinin
and discloses the partially cryptic binding region for PKN, although
the other possibility cannot be ruled out that PI4,5P2 functions as a
bridge between
-actinin and PKN. This binding activity was elevated
with increased PI4,5P2 concentration up to 2.5-10 µM and
then was lowered to 100 µM (Fig. 7B). This
two-phase pattern of PI4,5P2 dependence was also reported in the
binding of
-actinin with PI3-kinase (31). Fukami et al.
(30) reported that the effect of PI4,5P2 on gelating activity of
-actinin is increased up to 5-10 µM of PI4,5P2 and
that a further increase in concentration of PI4,5P2 gives a reduction
in gelating activity to the basal level due to the formation of large
PI4,5P2 micelles. The two-phase pattern of
PI4,5P2-dependent binding of
-actinin with PKN also may
be explained by the same reason.
Fig. 7.
Effects of PI4,5P2 on interaction of PKN with
-actinin. 35S-Labeled in vitro
translated full-length coding region of HuActSk1 was incubated with the
bacterially synthesized GST or GST-fused PKNN1. Aliquots of the initial
binding reaction mixtures (10 µl) were removed before precipitation
and applied to electrophoresis, which were indicated as
Input. GST or GST-fused proteins were collected with
glutathione-Sepharose beads, analyzed by 8% SDS-PAGE, and followed by
autoradiography. Arrow indicates the position of labeled
HuActSk1. A, specific binding of full-length
-actinin with PKN in the absence (lanes 1, 2, 5, and 6) or
presence (lanes 3, 4, 7, and 8) of 10 µM PI4,5P2. Lanes 1, 3, 5, and 7,
GST-PKNN1; lanes 2, 4, 6, and 8, GST.
B, effects of concentration of PI4,5P2 on the binding
activity of full-length
-actinin with PKN. Lanes 1 and
6, 0 µM PI4,5P2; lanes 2 and
7, 2.5 µM PI4,5P2; lanes 3 and
8, 10 µM PI4,5P2; lanes 4 and
9, 30 µM PI4,5P2; lanes 5 and 10, 100 µM PI4,5P2.
[View Larger Version of this Image (23K GIF file)]
-Actinin on the PKN Kinase Activity
-actinin to PKN directly altered
PKN regulation or catalytic function. The purified
-actinin from
bovine aorta neither activated PKN autophosphorylation nor affected
PKN-catalyzed protein kinase C pseudosubstrate peptide phosphorylation
when added at >100 molar excess to PKN (data not shown). When assayed
in the presence of 10 µM PI4,5P2, peptide phosphorylation
activity of PKN was stimulated ~1.5-fold; however, addition of
-actinin to this assay mixture slightly inhibited the
phosphorylation activity toward the basal level. Thus,
-actinin does
not seem to be a direct activator of PKN purified from the soluble
fraction of rat testis in vitro.
-Actinin and Other Actin
Cytoskeletal-associated Proteins by PKN
-actinin, we tested whether
-actinin itself could be a substrate
for PKN. In the absence of modifiers, PKN purified from rat testis did
not phosphorylate
-actinin purified from bovine aorta. However, in
the presence of 40 µM arachidonic acid, PKN
phosphorylated purified
-actinin with a stoichiometry of ~0.02 mol
of Pi per protein monomer by image quantitation (Fig. 8A). The bacterially expressed C-terminal
region of
-actinin (amino acid 333-894) was not phosphorylated at
all by PKN, but PKN phosphorylated the bacterially expressed N-terminal
region of
-actinin (amino acid 1-332) that was lacking in the
originally isolated clone 4 in the presence of arachidonic acid (data
not shown), suggesting that phosphorylation of
-actinin by PKN
occurred in the N-terminal region. We searched for PKN substrates among other actin cytoskeletal proteins, including filamin, metavinculin, vinculin, talin, caldesmon, and actin. Among them, caldesmon and G-actin were relatively preferred substrates for PKN. (The maximal phosphorylation by PKN per mol of protein subunit was estimated by
image quantitation to be ~0.3 mol of Pi per mol of
caldesmon and ~0.05 mol of Pi per mol of G-actin,
respectively.) As shown in Fig. 8B, phosphorylation of
G-actin and caldesmon was stimulated up to ~2-fold and >6-fold in
the presence of arachidonic acid, respectively.
Fig. 8.
Phosphorylation of actin and actin-binding
proteins by PKN. Phosphorylation was detected by an autoradiograph
of SDS-PAGE. White arrowhead indicates the position of
autophosphorylation of PKN. Molecular mass markers are indicated in
kDa. A, 100 ng of purified -actinin was incubated with
assay mixture without (lane 1) or with (lanes 2 and 3) PKN purified from rat testis in the absence
(lanes 1 and 2) or presence (lane 3)
of 40 µM arachidonic acid. Black arrowhead
indicates the position of
-actinin. B, 100 ng of purified
G-actin (lane 1-3) or caldesmon (lane 4-6) was
incubated in the assay mixture without (lanes 1 and
4) or with (lanes 2, 3, 5, and 6) PKN
purified from rat testis in the absence (lanes 2 and
5) or presence (lanes 3 and 6) of 40 µM arachidonic acid. Black arrow indicates the
position of caldesmon, and black arrowhead indicates the
position of G-actin.
[View Larger Version of this Image (34K GIF file)]
-actinin (30), profilin (40), gelsolin (41), cofilin (42), and
p39capZ (43). It has also been shown that the decrease in
PI4,5P2 bound to
-actinin and vinculin by treatment with
platelet-derived growth factor correlates with the depolymerization of
actin (44). Recently, Glimore and Burridge (45) have reported that
microinjection of antibodies against PI4,5P2 into Balb/c 3T3 cells
inhibits assembly of stress fibers and focal adhesions by serum
stimulation. One possibility is that PI4,5P2 synthesis could mediate
some of the effects of Rho on the actin cytoskeleton (45). In our
experiment, Rho directly interacts with and activates PKN, and PKN
could directly associate with
-actinin in
PI4,5P2-dependent manner in vitro. On the other
hand, phosphoinositides such as PI4,5P2 have been reported to affect
directly the kinase activity of PKN in vitro (46). Thus, the
possibility is raised that PKN is also implicated in mediating some
effects of Rho on the actin cytoskeleton.
-Actinin itself and some
actin-based cytoskeletal proteins such as actin and caldesmon serve as
the relatively preferred substrates for PKN, although the
stoichiometries were low in our in vitro assay condition.
Thus one might further speculate that PKN mediates the effects of Rho
and phosphoinositides by phosphorylating these proteins, although it is
not known whether these proteins are physiologically relevant
substrates of PKN. Further investigation will be required to clarify
the role of PKN in the cytoskeletal network.
*
This work was supported in part by research grants from the
Ministry of Education, Science, Sports and Culture, Japan, the Japan
Foundation for Applied Enzymology, and Kirin Brewery Co., Ltd. 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.
¶
To whom correspondence should be addressed: Dept. of Biology,
Faculty of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe
657, Japan. Tel.: (81-78)-803-0556; Fax: (81-78)-803-0322; E-mail:
yonodayo{at}icluna.kobe-u.ac.jp.
1
The abbreviations used are: HuActSk1, human
skeletal muscle type 1 -actinin; aa, amino acid or amino acids; GST,
glutathione S-transferase; HuActNm, human non-skeletal
muscle type
-actinin; PCR, polymerase chain reaction; DTT,
dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PI4,5P2,
phosphatidylinositol 4,5-bisphosphate; HA, hemagglutinin.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.