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(Received for publication, August 28, 1996, and in revised form, September 30, 1996)
From the Biosignal Research Center, Kobe University, Kobe 657, Japan, the § Department of Biology, Faculty of Science,
Kyushu University, Fukuoka 812-81, Japan, the ¶ Inheritance and
Variation Group, PRESTO, Research Development Corporation of Japan,
Kyoto 619-02, Japan, and the ABSTRACT The LIM domain comprising two zinc-finger motifs
is found in a variety of proteins and has been proposed to direct
protein-protein interactions. During the identification of protein
kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a
novel protein containing three LIM domains, designated ENH, was shown
to associate with PKC in an isoform-specific manner. Deletion analysis
demonstrated that any single LIM domain of ENH associates with the
NH2-terminal region of PKC. ENH associated with PKC in
COS-7 cells and was phosphorylated by PKC in vitro. Upon
treatment of the cells with phorbol ester, ENH in the membrane fraction
was translocated to the cytosol fraction in vivo. Other LIM
domain-containing proteins, such as Enigma and LIM-kinase 1, also
interacted with PKC through their LIM domains. These results suggest
that the LIM domain is one of the targets of PKC and that the LIM-PKC
interaction may shed light on undefined roles of LIM domain-containing
proteins.
INTRODUCTION The LIM domain is a Cys-rich domain composed of 50-60 amino
acid residues with the consensus sequence
(Cys-X2-Cys-X17-19-His-X2-Cys)-X2-(Cys-X2-Cys-X16-20-Cys-X2-His/Asp/Cys) (where X represents any amino acid) and is found in various
proteins (1, 2): homeodomain-containing transcription factors,
cytoskeletal proteins, LIM domain only proteins, protein kinases, and
proteins of undefined function. Physicochemical and structural analyses have revealed that the LIM domain is composed of two independent zinc-coordinated fingers (3, 4). Although many zinc finger motifs bind
to specific DNA or RNA sequences (5), the LIM domain has been proposed
to participate in protein-protein interactions (1, 2). In fact, five
proteins have been reported recently as a highly specific target of
each LIM domain: the LIM1 domain of zyxin binds to the LIM-only protein
CRP by LIM-LIM interaction (6); the CRP forms homodimer by LIM-LIM
interaction (7); the LIM domain of RBTN2 binds to the bHLH
(basic-helix-loop-helix) domain of TAL1 protein (8); the LIM2 and LIM3
domains of Enigma interact with the Tyr-containing tight-turn motifs of
the GDNF1 receptor (GDNFR, known as a Ret
Tyr kinase) and the insulin receptor (InsR), respectively (9, 10).
Although most LIM domains adopt a similar zinc-coordinated finger
consisting of well conserved amino acid sequences, no protein has been
identified yet as a common target of LIM domains.
The PKC family consists of at least 11 isoforms, which play distinct
roles for many cellular functions but show subtle difference of
substrate specificities by in vitro phosphorylation studies (11, 12). Therefore, it is reasonable to assume that there are some
mechanisms by which each PKC isoform recognizes its specific substrate
proteins in vivo. Recently, several proteins associating with PKC have been emerged to govern the subcellular localization of
the enzyme family (13, 14, 15, 16).
We report here a novel PKC-binding protein containing three LIM
domains, designated ENH, and show the association of PKC with LIM
domains of different proteins including this novel PKC-binding protein,
suggesting that protein-protein interaction with PKC is a general
property of LIM domains.
EXPERIMENTAL PROCEDURES The yeast two-hybrid assay (17) was
conducted in the yeast strain CG-1945 , a derivative of HF7c (18), by
using a fusion between GAL4 DNA binding domain and the regulatory
domain of rat PKC We
constructed two parental vectors, pTB701-FLAG and pTB701-HA, for
expression of NH2-terminal epitope-tagged fusion proteins in COS-7 cells, by inserting the sequences encoding FLAG (20) and HA
epitope (21) under the SV40 early promoter of pTB701 (22),
respectively. We constructed pTB701-FLAG-ENH by fusing the ENH cDNA
under the FLAG epitope sequence of pTB701-FLAG. Similarly, pTB701-HA-PKC COS-7 cells
coexpressing FLAG-tagged ENH and HA-tagged PKC The reaction mixture (25 µl) and 50 ng of PKC (mixture of RESULTS AND DISCUSSION We used a yeast two-hybrid system to identify proteins that bind
to the regulatory domain of PKC
To investigate the in vivo interaction between ENH and PKC
We next examined whether ENH is a substrate for PKC by incubating
bacterially expressed GST-ENH hybrid with purified rat cPKC. The
GST-ENH fusion protein was phosphorylated by PKC in vitro, whereas GST alone was not (Fig. 3B, top and bottom
panels). H1 histone, known as a good substrate for PKC (26), was
more efficiently phosphorylated than GST-ENH by PKC (Fig. 3B,
middle panel). H1 histone was known to be rapidly phosphorylated
by PKC, and after prolonged reaction approximately 1.8 mol of phosphate
was incorporated into every mole of H1 histone (26). Under similar
conditions, the reaction with GST-ENH was relatively slow. After
prolonged reaction, approximately 0.9 mol of phosphate was incorporated per mol of GST-ENH. These data suggest that ENH is a good substrate of
PKC at least under the conditions employed.
Western blotting of subcellular fractions of COS-7 cells coexpressing
ENH-FLAG and PKC We next delineated the region of ENH that interacts with the regulatory
domain of PKC
Because human Enigma (9) has three highly related LIM domains at its
COOH-terminal (Enigma LIM1-3 domain), we investigated the interaction
of Enigma LIM domains with PKC by a pull-down assay. Cell lysates from
the COS-7 cells expressing one of the HA-tagged PKCs (
By the pairwise alignment analysis of LIM domains, Dawid et
al. (1) demonstrated that LIM domains are classified into five discrete groups (groups A to E). All LIM domains of ENH and Enigma belong to group D. We tested LIM domains from other groups using human
LIM-kinase 1 (LIMK1) (30, 31), which harbors two LIM domains at its
NH2 terminus (LIM1 from group A, LIM2 from group B). In
COS-7 cells, we found that the LIMK1 interacts tightly with PKC In this study, we demonstrated that the LIM domains selectively bind to
PKC isoforms in vitro and in vivo. The LIM-PKC
interaction may be an important clue for the understanding of PKC
isoform-specific functions in vivo and the roles of LIM
domain-containing proteins.
FOOTNOTES Acknowledgments We thank Dr. Y. Nishizuka for encouragement
and M. Inagaki for secretarial assistance.
REFERENCES
Volume 271, Number 49,
Issue of December 6, 1996
pp. 31029-31032
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
,
and
Department of Medicine, University
of California San Diego, La Jolla, California 92093-0650
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
I (residues 1-340) (19) as a bait.
-Galactosidase activity in yeast cells was measured by plate assay
methods. All measurements were repeated at least four times.
I was constructed by inserting the PKC I cDNA to pTB701-HA. HA-tagged PKC 
, 
, 
, 
, and 
were
constructed using each insert cDNA (23, 24). A kinase-negative
mutant of PKC I-HA was generated by replacing Lys-371 by Met by
site-directed mutagenesis, and designated as K371M PKC I-HA.
I from a 10-cm plate
were suspended in 500 µl of lysis buffer (50 mM Tris, pH
7.5, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 10 mM 2-mercaptoethanol, 50 mM NaF, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 1 tablet/50 ml complete protease
inhibitor mixture tablets (Boehringer Mannheim), 1% (v/v) Triton
X-100). Cleared lysates (500 µl) were incubated for 1 h on ice
with 2 µg of either anti-FLAG (M2, Eastman Kodak Co.) or anti-HA
(12CA5, Boehringer Mannheim) monoclonal antibody and then mixed with 20 µl of protein G-Sepharose 4 fast flow (50% slurry, Pharmacia
Biotech, Uppsala, Sweden). After incubation at 4 °C for 1 h
with rotation, the beads were washed with lysis buffer four times. The
beads were subjected to Western blotting. For phosphorylation assay,
the beads were mixed with 25 µl of the reaction mixture (20 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM CaCl2, 20 µM ATP, 8 µg/ml
phosphatidylserine, 0.8 µg/ml diolein). After addition of 1 µl of
[-32P]ATP (10 mCi/ml), the reaction mixture was
incubated for 15 min at 30 °C. Samples were analyzed on SDS-PAGE and
then autoradiographed.
, I, II, ) purified from
rat brain (25) was added to 5 µg of GST-fused ENH bound to
glutathione-Sepharose 4B. After addition of 1 µl of
[-32P]ATP (10 mCi/ml), the reaction mixture was
incubated for 5 min at 30 °C. Samples were analyzed onto SDS-PAGE
and then autoradiographed.
I. Six positive clones were isolated
independently from a rat brain cDNA library. Sequence analysis
showed that one of the positive clones encodes a novel protein
containing LIM domains. A full-length cDNA clone (1,896 base pairs)
was obtained by a rapid amplification of cDNA ends method from the
same cDNA library (Fig. 1). The cDNA encodes a novel polypeptide sequence of 591 amino acid residues with a calculated molecular weight of 63,197. The deduced protein sequence has two Pro/Ser-rich regions (Pro/Ser-1 (residues 106-216): Pro, 18.9%; Ser,
19.8%; Pro/Ser-2 (residues 308-394): Pro, 16.1%; Ser, 23.0%) and
three LIM domains (LIM1-3; residues 415-585), which resembles the
molecular organization of human Enigma (455 amino acid residues) (9).
Residues 120-591 of this protein show high similarity to the
full-length Enigma, and approximately 37% of the amino acid residues
are identical between this protein and Enigma. Therefore, we termed
this protein ENH (
igma 
omolog). Northern
blot analysis of adult rat tissues revealed that the 1.9-kb ENH
mRNA was efficiently expressed in heart and skeletal muscle, and
the 4.4-kb ENH mRNA was expressed in various tissues, such as
heart, brain, spleen, liver, and kidney (Fig. 2).
Fig. 1.
Alignment of rat ENH and human Enigma amino
acid sequences. Sequence comparison was carried out using a BLOSUM
62 amino acid substitution matrix, and insertions (indicated with
dots) were introduced to optimize the alignment. Identical
and similar residues are indicated with bars and
pluses, respectively. Different residues are indicated with
minuses. Pro/Ser-rich region 1 (Pro/Ser-1) is indicated with
a thick line and Pro/Ser-rich region 2 (Pro/Ser-2) with a
double line. Three LIM domains (LIM1, -2, -3) are indicated with boxes. The numbers of amino acid residues
are shown on the left. The original clone isolated by
two-hybrid assay corresponds to amino acid residues Asn-302 to Tyr-520.
The sequence was deposited in GenBankTM with accession number
U48247[GenBank].
[View Larger Version of this Image (68K GIF file)]
Fig. 2.
Northern blot analysis of ENH mRNA.
Northern blot of polyadenylated mRNA isolated from various adult
rat tissues (Clontech) was used . The positions of ENH mRNA are
indicated on the right with arrowheads. The
positions of RNA standards (in kb) are indicated on the left
with arrows.
[View Larger Version of this Image (24K GIF file)]
I, we constructed expression vectors of FLAG-tagged ENH (ENH-FLAG) and HA-tagged PKC I (PKC I-HA). COS-7 cells coexpressing ENH-FLAG and PKC I-HA were lysed, and proteins were immunoprecipitated with
either anti-FLAG or anti-HA antibody. Western blotting analysis (Fig.
3A, top and bottom panels) showed
that ENH-FLAG (70 kDa) associated with PKC I-HA (80 kDa) in
vivo. TPA treatment prior to the cell lysis had no effect on the
association of these two proteins. Phosphorylation assay (Fig.
3A, middle panel) indicated that PKC I-HA can
phosphorylate ENH-FLAG. When a kinase-negative mutant of PKC I-HA
(K371M PKC I-HA) was used under the same condition (Fig.
3A), although the association of ENH-FLAG with the kinase
mutant was observed, the phosphorylation of ENH-FLAG was not observed.
These results indicate that association of PKC I with ENH is
independent of the enzymatic activity of PKC I.
Fig. 3.
Interaction of ENH and PKC I.
A, PKC I-HA and its kinase-negative mutant (K371M PKC
I-HA) interact with ENH-FLAG in COS-7 cells. Either PKC I-HA or
K371M PKC I-HA was coexpressed with ENH-FLAG in COS-7 cells. PKC
I-HA and K371M PKC I-HA (indicated with arrow in
top panel) in anti-FLAG immunoprecipitates was detected with
anti-HA antibody, and ENH-FLAG (indicated with arrow in
bottom panel) in anti-HA immunoprecipitates was detected
with anti-FLAG antibody. Anti-FLAG immunoprecipitates were subjected to
phosphorylation assay (middle panel). ENH-FLAG (indicated
with arrow) was phosphorylated by PKC I-HA, not by
K371M PKC I-HA. B, phosphorylation of GST-ENH by PKC
in vitro. GST-ENH (5 µg), H1 histone (5 µg), and GST (10 µg) (indicated with arrows) were phosphorylated with
purified rat PKC at 30 °C for 5 min, subjected to SDS-PAGE, stained
with Coomassie Brilliant Blue R-250 (left panels), and
autoradiographed (right panels). C, subcellular
localization of ENH-FLAG in COS-7 cells. Cells coexpressing PKC I-HA
and ENH-FLAG were treated with TPA (100 nM) for 15 min at
37 °C. Where indicated calphostin C (100 nM) was added
for 15 min at 37 °C prior to TPA treatment. Cells were fractionated
by standard procedures. ENH-FLAG from the cytosol fraction (top
panel) and the membrane fraction (bottom panel)
(indicated with arrows) was analyzed with anti-FLAG
antibody. Each lane contains the sample derived from approximately
5 × 105 cells. Based on the densitometric intensity
of each band, the relative amount of ENH-FLAG in each lane was
indicated below (the amount of ENH-FLAG in the cytosol of untreated
cells is defined as 1.0).
[View Larger Version of this Image (31K GIF file)]
I-HA showed that ENH-FLAG is equally localized in
both cytosol and membrane fractions (Fig. 3C). After TPA
treatment, ENH-FLAG in the membrane fraction disappeared, and the
amount of ENH-FLAG in the cytosol fraction increased. When a PKC
inhibitor, such as calphostin C (100 nM) (27), was added
for 15 min prior to TPA treatment (Fig. 3C), ENH-FLAG
remained in the membrane fraction. Staurosporine (100 nM),
a general inhibitor of Ser/Thr protein kinases (28), also blocked the
TPA-induced translocation of ENH. When we used ENH-HA instead of
ENH-FLAG, the same results were obtained. Thus, ENH interacts with PKC
I in vivo, and activation of PKC results in translocation
of ENH from membrane to cytosol.
I by the yeast two-hybrid system. A series of deletion
mutants of ENH as GAL4 DNA binding domain hybrids were examined for the
interaction with the regulatory domain of PKC I (Fig.
4A). The NH2-terminal region of
ENH (residues 1-414; 
LIM) is not required for PKC binding, whereas
each LIM domain (LIM1, LIM2, LIM3) can bind to PKC I. In addition,
the COOH-terminal half of the LIM1 domain (1/2 LIM1) was unable to
interact with PKC I. These results indicate that the region of ENH
essential for the PKC I binding is a single intact LIM domain. Using
the regulatory domain of various PKCs as a bait, it was shown that residues 415-591 of ENH (ENH LIM1-3 domain) were able to interact with PKC I, , and but not with PKC , , and . To
further delineate the region interacting with the ENH LIM domains, we assayed deletion mutants of the regulatory domain of PKC I and (Fig. 4B). Mutants harboring the NH2-terminal V1
region of either PKC I or showed an intact ability to bind to
the ENH LIM1-3 domain. The V1 region of PKC is thus critical for the
ENH-PKC interaction. Since the V1 regions of PKCs show high diversity (29), it is likely that the LIM domains of ENH may identify PKC
isoforms by a specific sequence of the V1 region rather than the
conformation of PKC molecule.
Fig. 4.
The interaction of ENH and PKC in the yeast
two-hybrid system. -Galactosidase activity of yeast
transformants was assayed by the plate method (17). After developing
for 10 h, the yeast cells were classified into positive colonies
showing either dark blue (++) or blue (+), and negative colonies
showing white (
). A, delineation of the domain of ENH which interacts with the regulatory domain of PKC I. P/S1, P/S2, 1, 2,
and, 3 in the schema represent Pro/Ser-rich region 1, Pro/Ser-rich region 2, LIM1, LIM2, and LIM3, respectively.
B, delineation of the domain of PKC I and which
interacts with the ENH LIM1-3. Based on the sequence similarity, the
primary structure of the regulatory domain of PKC I and is
divided into conserved domains (boxes), which are separated
by variable regions (lines) (29).
[View Larger Version of this Image (10K GIF file)]
, I, ,
, , and ) were mixed with GST or GST-Enigma LIM1-3, and the
proteins bound to glutathione-Sepharose 4B were analyzed with anti-HA
antibody. The Enigma LIM1-3 domain was found to interact with PKC ,
I, and , but not with PKC , , and (Fig.
5A, top panel), indicating that LIM domains of Enigma and ENH have individual specificities for PKC isoforms. Previous studies showed that the LIM2 and LIM3 domains of Enigma bind
to GDNFR and InsR, respectively (9, 10). Each LIM domain of Enigma was
found to associate with PKC, as in the case of ENH. These results
suggest the possibility that one LIM domain interacts with more than
one molecule. Enigma has been postulated to be either a scaffold or an
anchoring protein that coordinates the subcellular localization and
activity of GDNFR and InsR (9, 10) and also associates with PKC as
described above. Since ENH shows the same molecular organization as
Enigma, ENH might have a role similar to Enigma and its subcellular
localization is controlled by PKC.
Fig. 5.
The interaction of LIM domains of Enigma and
LIM-kinase 1 with PKC. A, the interaction of Enigma LIM
domains with PKC in vitro. Cell lysates used were prepared
from COS-7 cells expressing PKC-HA (, I, , , , and ).
The positions of PKC -HA (approximately 80 kDa) in the blots were
indicated with arrows on the left. B, the interaction of LIMK and PKC in vivo. The interactions
between LIMK1-FLAG and PKC-HA were detected by the immunoprecipitation assay (bottom panel). Cell lysates used were prepared from
COS-7 cells coexpressing PKC-HA and LIMK1-FLAG. The positions of
LIMK1-FLAG (approximately 73 kDa) and PKC -HA (approximately 80 kDa)
in the blots were indicated with closed and open
arrows, respectively. The contents of LIMK1-FLAG and PKC-HA in the
immunoprecipitates were monitored by Western blotting (top
and middle panels). C, interaction of PKC -HA
and LIMK mutants in vivo. LIMK1(1m), carrying mutated LIM1 and native LIM2 domain; LIMK1(2m), carrying
native LIM1 and mutated LIM2 domain; and LIMK1(dm), carrying
mutated LIM1 and mutated LIM2 domain. Each full-length LIMK1 mutant and PKC -HA were coexpressed in COS-7 cells. The interaction of PKC -HA and each LIMK1 mutant was analyzed with anti-HA (third
panel) and anti-LIMK1 antibody (fourth panel). The
positions of LIMK1 mutants (approximately 73 kDa) and PKC -HA
(approximately 80 kDa) in the blots were indicated with
closed and open arrows, respectively. The
contents of LIMK1 mutants and PKC -HA in the immunoprecipitates were
monitored by Western blotting (first and second
panels).
[View Larger Version of this Image (41K GIF file)]
and
, and weakly with PKC, I, , and (Fig. 5B, bottom
panel). To confirm that the LIMK1-PKC interaction is mediated by
the LIM domains of LIMK1, we constructed expression vectors for three
LIMK1 mutants, to which LIM domains were mutated not to form zinc
binding by substituting Gly for consensus Cys (32), see Fig.
5C. COS-7 cells coexpressing each LIMK1 mutant and PKC -HA, which efficiently bound to LIMK1, were lysed, and proteins were
immunoprecipitated with either anti-LIMK1 (30, 31) or anti-HA antibody.
Western blotting (Fig. 5C, third and fourth panels) indicated that the LIM2 domain of LIMK1 (group B) is
critical for the binding between LIMK1 and PKC . Further analysis of
other groups of LIM domains should yield insight into the general role of the LIM-PKC interaction.
To whom correspondence should be addressed: Biosignal Research
Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657, Japan.
Tel.: 81-78-803-1255; Fax: 81-78-803-0994; E-mail:
skuroda{at}inherit.biosig.kobe-u.ac.jp.
1
The abbreviations used are: GDNF,
glial-cell-line-derived neurotrophic factor; GDNFR, GDNF receptor;
InsR, insulin receptor; PKC, protein kinase C; PAGE, polyacrylamide gel
electrophoresis; kb, kilobases; TPA,
12-O-tetradecanoylphorbol-13-acetate; LIMK1, LIM-kinase 1;
GST, glutathione S-transferase.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. Muto, J. Ruland, L. M. McAllister-Lucas, P. C. Lucas, S. Yamaoka, F. F. Chen, A. Lin, T. W. Mak, G. Nunez, and N. Inohara Protein Kinase C-associated Kinase (PKK) Mediates Bcl10-independent NF-kappa B Activation Induced by Phorbol Ester J. Biol. Chem., August 23, 2002; 277(35): 31871 - 31876. [Abstract] [Full Text] [PDF]
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 T. Vallenius and T. P. Makela Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein J. Cell Sci., May 15, 2002; 115(10): 2067 - 2073. [Abstract] [Full Text] [PDF]
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Q. Zhou, P.-H. Chu, C. Huang, C.-F. Cheng, M. E. Martone, G. Knoll, G. D. Shelton, S. Evans, and J. Chen Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy J. Cell Biol., November 12, 2001; 155(4): 605 - 612. [Abstract] [Full Text] [PDF]
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 D. Mehta Serine/threonine phosphatase 2B regulates protein kinase C-{alpha} activity and endothelial barrier function Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L544 - L545. [Full Text] [PDF]
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 K. Bauer, M. Kratzer, M. Otte, K. L. de Quintana, J. Hagmann, G. J. Arnold, C. Eckerskorn, F. Lottspeich, and W. Siess Human CLP36, a PDZ-domain and LIM-domain protein, binds to alpha -actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells Blood, December 15, 2000; 96(13): 4236 - 4245. [Abstract] [Full Text] [PDF]
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 H. Tanahashi and T. Tabira Alzheimer's disease-associated presenilin 2 interacts with DRAL, an LIM-domain protein Hum. Mol. Genet., September 1, 2000; 9(15): 2281 - 2289. [Abstract] [Full Text] [PDF]
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T. Vallenius, K. Luukko, and T. P. Makela CLP-36 PDZ-LIM Protein Associates with Nonmuscle alpha -Actinin-1 and alpha -Actinin-4 J. Biol. Chem., April 6, 2000; 275(15): 11100 - 11105. [Abstract] [Full Text] [PDF]
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