(Received for publication, August 24, 1995; and in revised form, November 17, 1995)
From the
We previously reported that phosphatidylinositol
4,5-bisphosphate (PIP) dramatically increases the gelating
activity of smooth muscle
-actinin (Fukami, K., Furuhashi, K.,
Inagaki, M., Endo, T., Hatano, S., and Takenawa, T.(1992) Nature 359, 150-152) and that the hydrolysis of PIP
on
-actinin by tyrosine kinase activation may be important in
cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and
Takenawa, T.(1994) J. Biol. Chem. 269, 1518-1522). Here
we report that a proteolytic fragment with lysylendopeptidase
comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated
muscle
-actinin contains a PIP
-binding site. A
synthetic peptide composed of the 17 amino acids remarkably inhibited
the activities of phospholipase C (PLC)-
1 and -
1.
Furthermore, we detected an interaction between PIP
and a
bacterially expressed
-actinin fragment (amino acids
137-259) by PLC inhibition assay. Point mutants in which arginine
172 or lysine 184 of
-actinin were replaced by isoleucine reduced
the inhibitory effect on PLC activity by nearly half. Direct
interactions between PIP
and the peptide (amino acids
168-184) or the bacterially expressed protein (amino acids
137-259) were confirmed by enzyme-linked immunosorvent assay. We
also found this region homologous to the sequence of the
PIP
-binding site in spectrin and the pleckstrin homology
domains of PLC-
1 and Grb7. Synthetic peptides from the homologous
regions in spectrin and PLC-
1 inhibited PLC activities. These
results indicate that residues 168-184 comprise a binding site
for PIP
in
-actinin and that similar sequences found
in spectrin and PLC-
1 may be involved in the interaction with
PIP
.
Phosphatidylinositol 4,5-bisphosphate (PIP) (
)is a trace phospholipid, which generates two second
messengers, inositol 1,4,5-trisphosphate and diacylglycerol that
respond to phospholipase C (PLC) activation by a variety of
physiological stimuli. Inositol 1,4,5-trisphosphate and diacylglycerol
are known to mobilize Ca
from the endoplasmic
reticulum and to activate protein kinase C (PKC),
respectively(3, 4) .
In addition to its role as a
signal-generating lipid, PIP has been shown to modulate the
functions of various proteins such as PKC(5, 6) ,
µ-calpain(7) , ADP-ribosylation factor 1(8) , and
phospholipase D(9) . PIP
also binds to
actin-regulating proteins such as profilin(10) ,
cofilin(11) , gelsolin(12) , gCap(13) , and
-actinin (1) and regulates the functions of these
proteins. When PIP
binds to
-actinin, which is an
actin cross-linking protein, it further activates actin gelation by
-actinin(1) . It is noteworthy that profilin plays crucial
roles in tyrosine kinase-coupled PIP
hydrolysis. Under
resting conditions, PLC-
1 causes little hydrolysis of
profilin-bound PIP
, but PLC-
1 phosphorylated by
tyrosine kinases overcomes the inhibitory effect by profilin and
hydrolyzes bound PIP
(14) . It has also been shown
that the decrease in PIP
bound to
-actinin and
vinculin by treatment with platelet-derived growth factor correlates
with the depolymerization of actin(2) . All these data suggest
that the amount of PIP
in the actin-binding protein
regulates the development of stress fibers when the cells are
stimulated.
-Actinin was originally discovered in skeletal
muscle as a protein factor promoting the superprecipitation of
actomyosin and inducing the formation of actin fibers(15) . The
fact that
-actinin is found at focal contacts where actin is
anchored to a variety of intercellular structures in non-muscle cells
suggests that
-actinin plays some role in the linkage between the
plasma membrane and actin. We previously reported that
-actinin
from skeletal muscle contains large amounts of PIP
, whereas
that from smooth muscle contains little(1) . Interestingly, the
addition of PIP
to smooth muscle
-actinin increases
the gelation activity of actin to the level produced by skeletal muscle
-actinin, suggesting that PIP
plays important roles in
the organization of the cytoskeleton.
Recently, the preckstrin
homology (PH) domain has been found in a variety of functional
proteins(16) , including protein kinases, substrates for
kinases, regulators of small G proteins, PLC isozymes, and cytoskeletal
proteins. This domain has been reported to bind to
PIP(17) , although it also associates with the
subunit of trimeric G proteins (18, 19) and
PKC(20) . In that case, PIP
is thought to act as a
target for PH domain-containing proteins in membranes.
To understand
the role of PIP in protein functioning or in
protein-protein interactions, it is important to identify the
PIP
-binding site in proteins. We describe here that amino
acids 168-184 in chicken skeletal muscle
-actinin comprise a
PIP
-binding site and that basic amino acids, arginine 172
and lysine 184, are important for this interaction. A region homologous
to the PIP
-binding site in
-actinin is also found in
spectrin and the PH domains of several proteins including PLC-
1
and Grb7.
Cleavage with lysylendopeptidase was carried out as
follows. -Actinin was digested overnight with 1:200 (mol/mol)
lysylendopeptidase in 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM 2-mercaptoethanol, and 1 M urea at 37
°C. The digests were separated by high performance liquid
chromatography on a C18 reverse-phase column. For the detection of
PIP
-bound peptide, every fraction, including the front, was
lyophilized, dot-blotted on nitrocellulose, and stained with
anti-PIP
antibody. The amino acid sequence of the
PIP
-containing fragment was determined with a protein
sequencer (ABI 477A/120A).
Three more peptides corresponding to the
homologous sites of several proteins were synthesized. Human spectrin
-chain (PEP-spectrin), TAGYPHVNVTNFTSSWK; PLC-
1
(PEP-PLC-
1), LKGSQLLKVKSSSWR; and Grb7 (PEP-Grb7),
QLRGSGRGSGRKLWK. We also synthesized a peptide which is known as
PIP
-binding site in gelsolin (24) (PEP-gelsolin),
KLFQVKGRR.
Figure 1:
-Chymotryptic cleavage pattern of
chicken skeletal
-actinin. 50 µg of skeletal muscle
-actinin was digested with
-chymotrypsin (molar ratio, 1:200)
at 37 °C for the indicated times as described under
``Experimental Procedures.'' The digests were subjected to
8.5% acrylamide gels and stained with Coomassie Brilliant Blue (A) or transferred to nitrocellulose and immunostained with
anti-PIP
antibody (B). The sizes of the
-chymotryptic cleavage fragments as reported previously (29) are shown in C.
To more closely locate the PIP-binding site,
limited proteolysis with lysylendopeptidase was carried out. The
digests of
-actinin were separated on a C18 reverse-phase column (Fig. 2A) and binding ability to PIP
was
assayed by dot-blot analysis with anti-PIP
antibody (Fig. 2B). Two positive peaks (shown by the arrowhead and * in Fig. 2A) were obtained. The
later peak (*), which was very broad and weakly positive for
PIP
, was found to be the 34-kDa N-terminal domain by
SDS-polyacrylamide gel electrophoresis (data not shown). On the other
hand, the earlier peak was very sharp and gave a very strong positive
signal for PIP
binding. We found the sequence of this
peptide to be TAPYRNVNIQNFHLSWK, which corresponds to the sequence of
amino acid residues 168-184 in chicken skeletal
-actinin. We
conclude therefore that this region in the actin-binding domain
contains a binding site for PIP
.
Figure 2:
Determination of the peptide sequence in
-actinin involved in PIP
binding by digestion with
lysylendopeptidase. 100 µg of
-actinin was treated overnight
with 0.38 µM lysylendopeptidase at 37 °C. The digests
were applied to a C18 reverse-phase column and eluted with 0-60%
gradient of acetonitrile (A). The separated peptides were
lyophillized and dot-blotted on nitrocellulose membranes. The membrane
was stained with anti-PIP
antibody (B). The
peptide containing PIP
was sequenced by a protein
sequencer.
A computer-assisted
sequence homology search revealed that this sequence is homologous to
chicken smooth muscle -actinin (88.8%), human spectrin
-chain
(58.8%), yeast mRNA capping protein (37.5%), and mouse integrin
-7
subunit precursor (35.7%). Moreover we found the homologous regions in
the PH domains of several proteins including PLC-
1, Grb 7,
pleckstrin, Ras-GAP, and racK
(Fig. 3).
Figure 3:
Comparison of the PIP-binding
domain of chicken striated
-actinin with various protein
sequences. Highly conserved (&cjs2110;) and similar (&cjs2106;) amino
acids residues are boxed and shaded, respectively. SK.
-actinin, chicken skeletal muscle
-actinin; SM.
-actinin, chicken smooth muscle
-actinin; spectrin, human spectrin
-chain; integrin, mouse
integrin
7 subunit precursor; mRNA.CP, yeast mRNA capping
protein; PLC
1.PH, Grb7.PH, plec(N).PH, plec(C).PH, Ras-GAP.PH, and racK
.PH are the PH domains of various proteins.
Amino acids alignment was done on the basis of
RXXXXXXX(H/R/K)XX(X)W(K/R).
Figure 4:
Inhibition of PLC-1 and PLC-
1
activities by synthetic peptides and
-actinin. Synthetic peptide
sequences are shown in A. The inhibition of PLC-
1 and
PLC-
1 activities by peptide I (
), peptide II (
),
peptide III (
), and peptide IV (
) at various doses is shown
in B. PLC activities were measured as described under
``Experimental Procedures'' using 20 µM PIP
as a substrate. The total activity of PLC-
1
is 5400 dpm and that of PLC-
1 is 8600 dpm. Inhibition by peptide
IV (
) in the presence of 0.5% octyl glucoside was also examined (B). C shows the inhibition of PLC-
1 and
PLC-
1 activities by smooth muscle
-actinin at concentrations
of 0, 3.8, and 19.1 µM. Inhibition of PLC-
1 and
PLC-
1 by peptides corresponding to PEP-spectrin (
),
PLC-
1 (
), PEP-Grb7 (
), and PEP-gelsolin (
) is
shown in D.
Figure 5:
Inhibition of PLC-1 and PLC-
1
activities by bacterial expression of PIP
-binding sites on
-actinin. Activities of PLC-
1 (A) and PLC-
1 (B) were measured in the presence of 20 µM various point mutants or a non-mutant as described under
``Experimental Procedures.''
Figure 6:
Direct interactions of PIP with peptides and bacterial expression
-actinin fragments.
96-well multiplates were coated with 50 µl of 10 µM peptide I (
), peptide II (
), peptide III (
),
peptide IV (
), or no peptide (&cjs0800;) (A), and 50
µl of 20 µg/ml
An.0.1 (
),
An.166.1 (&cjs0800;),
An.172.2 (
),
An.184.2 (circo]), or
An.195.1
(
) (B) overnight at room temperature. Various amounts of
PIP
were added to each well and incubated at room
temperature for 30 min. Procedures were the same as described under
``Experimental Procedures.''
There are many reports of specific interactions between
phospholipids and proteins. The C2 domains of
PKC(5, 6) , phospholipase A(28) ,
PLC, Ras-GTPase activating protein(29) , rabphilin (30) , and synaptotagmin I (31, 32) have been
proposed to contain phospholipid binding domains. ADP-ribosylation
factor I(8) , dynamin(33) , myristoylated alanine-rich
protein kinase C substrate (34) , µ-calpain(7) ,
and many actin-regulating
proteins(1, 10, 11, 12, 13, 14) have also been shown to interact with acidic phospholipids
including PIP
. These interactions induce the translocation
of PKC, synaptotagmin I, and dynamin to the plasma membrane, or
activate phospholipase D, ADP-ribosylation factor I, and µ-calpain.
Synaptotagmin I is thought to be involved in the docking and fusion
steps in calcium-dependent exocytosis. Interestingly, it has become
clear that PIP
synthesis by phosphatidylinositol
4-phosphate 5-kinase is also concerned in exocytosis(35) .
Additional evidence for a role of PIP
in vesicular
trafficking was provided by Cantley et al.(36) . They
reported that PIP
stimulates in vitro the activity
of partially purified membrane phospholipase D, in which PIP
functions as a phospholipase D cofactor(9) . These
results suggest that phospholipids by themselves play important roles
in modulating enzyme activities and targeting for translocation.
We
have shown that -actinin from chicken striated muscle contains
large amounts of PIP
while
-actinin from chicken
smooth muscle has little PIP
, but that the latter can bind
to exogenous PIP
. In vitro, the addition of
PIP
dramatically stimulates the gelating activity of actin
by smooth muscle
-actinin(1) . Furthermore, it has been
shown that the amount of PIP
bound to
-actinin and
vinculin decrease in response to platelet-derived growth factor
stimulation in vivo(2) . These facts suggest that
-actinin-bound PIP
is dynamically metabolized under
physiological conditions and that PIP
by itself regulates
the organization of stress fibers. Thus, we tried to clarify the
binding site of PIP
in
-actinin.
Amino acid
sequences which contain PIP-binding site in skeletal muscle
-actinin are homologous to that in chicken smooth muscle
-actinin (Fig. 3), except for the substitution of a basic
amino acid, arginine, to another basic amino acid, lysine. This
substitution may have no effect on PIP
-binding, but these
basic amino acids seem to be very important for binding, because
mutants in which either arginine 172 or lysine 184 is replaced by
isoleucine partially lose their inhibitory effect on PLC activities (Fig. 5) and their direct binding with PIP
(Fig. 6B). Sequences homologous to the
PIP
-binding domain in
-actinin also exist in some
cytoskeletal-related proteins such as spectrin
-chain or integrin
-7 subunit precursor, although these are not yet reported as
PIP
-binding proteins. On the other hand, we found no
homologous sequence in gelsolin or cofilin, which have been reported
previously to be PIP
-binding
proteins(11, 12) . For gelsolin, cofilin, and
profilin, we could detect no PIP
binding by Western blot
analysis. This may be due to the low affinity of PIP
for
these proteins compared to
-actinin.
We also found homologous
regions in the PH domains of several proteins. The PH domain is
suggested to be involved in protein-protein or lipid-protein
interactions, because the PH domain is reported to associate not only
with PIP(17) , but also with the
subunit
of trimeric G protein (18, 19) and PKC(20) .
An arginine to cysteine substitution in the N-terminal PH domain
(
-sheet) of Bruton's tyrosine kinase is thought
to be the cause of X-linked immunodeficiency in
mice(37) . This result suggests that the basic amino acid
arginine in the PH domain may play a critical role in the signaling of
Bruton's tyrosine kinase. Regions homologous to the
PIP
-binding site in
-actinin also exist in the
- and
-sheets of the PH domains of
PLC-
1 and Grb7. There has been another report that inositol
1,4,5-trisphosphate binds to PLC-
1 and that this interaction is
inhibited by PIP
(38) . The inositol
1,4,5-trisphosphate binding site on PLC-
1 is thought to comprise
amino acids 30-43, which overlaps with the site which we aligned
(amino acids 23-37). In fact, the peptide from PLC-
1
strongly inhibited the activity of PLC-
1, but PEP-Grb7 did not
inhibit. Although we do not know the precise reason, three-dimensional
conformation may be important for the interaction of the peptide and
PIP
.
We have shown that -actinin, a
bacterially-expressed protein, and a synthetic peptide corresponding to
amino acids 168-184 of
-actinin inhibit the activities of
PLC-
1 and PLC-
1. From these results, it appears likely that
PLC inhibition is induced by PIP
competition.
Goldschmidt-Clermont et al.(14) have reported that PLC-
1
causes little hydrolysis of PIP
bound to profilin, but that
PLC-
1 phosphorylated by tyrosine kinase overcomes the inhibitory
effect of profilin. Additionally, PIP
bound to
-actinin may be hydrolyzed by activated PLC-
1 when cells are
activated. This problem remains to be solved in future.