1 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and 2 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Representational difference analysis of
the glomerular endothelial cell response to transforming growth
factor-1 (TGF-
1) revealed a novel gene, TIMAP (TGF-
-inhibited
membrane-associated protein), which contains 10 exons and maps to human
chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed
in all cultured endothelial and hematopoietic cells. The frequency of
the TIMAP SAGE tag is much greater in endothelial cell SAGE databases
than in nonendothelial cells. Immunofluorescence studies of rat tissues
show that anti-TIMAP antibodies localize to vascular endothelium.
TGF-
1 represses TIMAP through a protein synthesis- and histone
deacetylase-dependent process. The TIMAP protein contains five ankyrin
repeats, a protein phosphatase-1 (PP1)-interacting domain, a
COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3,
and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein
localized to the plasma membrane in a CAAX box-dependent fashion.
Hence, TIMAP is a novel gene highly expressed in endothelial and
hematopoietic cells and regulated by TGF-
1. On the basis of its
domain structure, TIMAP may serve a signaling function, potentially
through interaction with PP1.
hematopoietic cells; rat; representational difference analysis; glomerular endothelial cells; human; vascular endothelial cells
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INTRODUCTION |
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MEMBERS OF THE
transforming growth factor- (TGF-
) family of homodimeric
extracellular ligands, which include TGF-
s, activins, and bone
morphogenetic proteins, regulate many cellular functions related to
development and differentiation (23, 24).
Cell signaling by the ligands of the TGF- superfamily occurs through
activation of cell surface receptor serine/threonine kinases and
subsequent activation of intracellular signaling molecules, the Smads
(6, 23). TGF-
1, the most studied member of the mammalian TGF-
s, binds directly to type II TGF-
receptor, a constitutively active receptor serine/threonine kinase. This
interaction also requires an accessory cell surface proteoglycan,
either
-glycan (20) or endoglin (4), the
latter expressed predominantly in endothelial cells. On TGF-
1
binding, recruitment of type I TGF-
receptors into a heteromeric
complex with type II receptors results in phosphorylation of type I
receptors and activation of their kinase activity. Transient
association of receptor-specific Smad2 and/or Smad3 (R-Smads) with
active type I receptors causes R-Smad phosphorylation, which is
followed by the formation of a heteromeric complex between R-Smad and
the related protein Smad4 (Co-Smad). The complex then moves to the
nucleus, where it regulates transcription by interacting with
cooperating transcriptional factors and Smad-responsive promoter
elements. It is now understood that the R-Smad-Co-Smad complex can
interact with diverse transcriptional regulators, for instance,
transcriptional factors interacting with activator protein-1 sites
(18, 43), transcriptional cofactors, such as FAST
(44), and transcriptional repressors, such as Ski, SnoN,
and TGIF (34, 40, 41).
Members of the TGF- superfamily are critical in controlling cell
proliferation, matrix synthesis, adhesion to matrix, and apoptosis, and absence of some components of the TGF-
pathway promotes tumor formation (22, 23, 37). The
specific effect of TGF-
1 and its family members is highly cell
dependent (24). In cultured endothelial cells, TGF-
1
inhibits cell proliferation (36), alters endothelial
cell-matrix interactions (28), and induces microvascular
endothelial cell apoptosis (5), an effect that can
be rescued by activation of integrin-
1
(29). In mice, deficiency of type II TGF-
receptor
(27) or endoglin, the class III TGF-
coreceptor
expressed by endothelial cells (17), results in an
embryonic lethal phenotype with aberrant vascular morphogenesis in the
yolk sac. Deficiency of ALK-1, a type I TGF-
receptor, also lethal
to embryos, has a very similar phenotype (26). The findings in humans that mutations in endoglin (25) and
ALK-1 (11) result in structural abnormalities of the
vasculature with the phenotype of hereditary hemorrhagic telangiectasia
also suggest that TGF-
signaling processes are critical in the
development and maintenance of normal vascular structures.
We previously reported that TGF- plays a critical role in glomerular
capillary morphogenesis. TGF-
1 stimulates assembly of cultured
glomerular endothelial (GEN) cells into capillaries, with the remainder
of the cells undergoing apoptosis (5). Capillary formation by GEN cells in vitro is abrogated in cells expressing the
dominant-negative type II TGF-
receptor and also by neutralizing TGF-
1 antibody. In vivo, normal glomerular capillary formation and
differentiation during renal development in rats are also dependent on
TGF-
1 (19).
Although there is ample evidence that TGF-1 is involved in the
formation and maintenance of blood vessel architecture, the molecules
downstream from the TGF-
1 signaling cascade in endothelial cells are
only partially understood. We therefore sought to identify previously
unknown targets of the TGF-
signaling cascade in endothelial cells.
Here we report the characterization of a novel gene, TIMAP (TGF-
-inhibited membrane-associated protein), highly expressed in
endothelial cells, which is strongly repressed by TGF-
1. The protein
product of this transcript contains several ankyrin repeats in its
NH2-terminal half and a COOH-terminal CAAX box, which
mediates its plasma membrane association.
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MATERIALS AND METHODS |
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Cell lines, culture, and cell treatment. Primary bovine GEN and bovine aortic endothelial (BAE) cells were prepared as described previously (1, 2). U-937 [histiocytic lymphoma, macrophage-like cell line; CRL-1593.2, American Type Culture Collection (ATCC)], human erythrocytic leukemia (HEL; TIB-180, ATCC), lymphoblastic T cell leukemia (MOLT4; CRL-1582, ATCC), human embryonic kidney (HEK-293; CRL-1573, ATCC), human erythroleukemia (TF-1; CRL-2003, ATCC), and Dami (a human megakaryocytic cell line; obtained from Dr. Paul F. Bray, Baylor University School of Medicine, Baylor, TX) cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS). Granulocyte/macrophage colony-stimulating factor (1 ng/ml) was added to TF-1 cell medium. KG-1a (human acute myelogenous leukemia, myeloblast; CCL-246.1, ATCC) and K-562 (human chronic myelogenous leukemia, lymphoblast; CCL-243, ATCC) cells were cultured in Iscove's modified Dulbecco's medium with 10% FBS. WM793 (a human melanoma cell line; obtained from Dr. Paul F. Bray), COS-7 (African green monkey kidney cell; CRL-1651, ATCC), and Madin-Darby canine kidney (MDCK; CCL-34, ATCC) cells were cultured in DMEM with 10% FBS. HeLa cells (human cervical carcinoma epithelial cell line; CCL-2, ATCC) were cultured in Eagle's minimum essential medium with 10% FBS. HCT116 cells (human colorectal carcinoma epithelial cell line; CCL-247, ATCC) were cultured in McCoy medium with 10% FBS. Human aorta endothelial cells, human umbilical vein endothelial cells, and human microvascular endothelial cells (HMEC-1) were purchased from Clonetics (San Diego, CA) and cultured in appropriate medium (Clontech) supplemented with 10% FBS, 10 µg/ml hydrocortisone (Sigma, St. Louis, MO), and 10 ng/ml epidermal growth factor (Collaborative Biomedical Products-Becton Dickinson, Bedford, MA).
In experiments delineating the TGF-Representational difference analysis.
For representational difference analysis (RDA), cDNA was synthesized
using poly(A)+ RNA template from GEN cells treated with
TGF-1 (GEN/TGF-
1 positive) or left untreated (GEN/TGF-
1
negative). The cDNA pools representing each cell population were
digested with DpnII, ligated to an oligonucleotide linker
with a 5' overhang [5'-AGCACTCTCCAGCCTCTCACCG CA-3' (R-24) and 5'-GATC TGCGGTGA-3' (R-24); complementary region for
the pair is underlined], and amplified with R-24 primer to generate
GEN/TGF-
1-positive and GEN/TGF-
1-negative amplicon pools. The
linkers were then removed from the amplicon cDNAs by DpnII
digestion and purification with Amicon 100 columns, generating
"driver" cDNAs. "Tester" cDNA representing each cell population
was generated by ligation of a distinct oligonucleotide linker with a
5' overhang [J-linker: 5'-ACCGACGTCGAC TATCCATGAACA-3'
(J-24) and 5'-GATCTGTTCATG-3' (J-12)] to a portion of each
driver pool.
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Full-length TIMAP cDNAs.
The initial 278-bp fragment of bovine TIMAP (bTIMAP) cDNA generated by
RDA was extended by 0.9 kb in the 3' direction using 3'-rapid
amplification of cDNA ends (RACE) with mRNA from GEN cells using a
Marathon 5'/3'-RACE kit (Invitrogen) and oligo 5'-CCAGATGCCCAGCTC CTGGTTAGA-3' as the 5' primer. The 1.1-kb 3'-RACE product was subcloned
into the pCR2.1 TA cloning vector and sequenced, and a BAE cell
ZAPII cDNA library (936705, Stratagene, La Jolla, CA) was then
screened by using the 1.1-kb TIMAP cDNA fragment as a probe.
From 106 plaque-forming units, two clones were isolated
that extended the sequence 4.3 kb in the 5' direction. A 693-bp
fragment from the 5' end of one clone was then used to screen the
library for a second time. Several overlapping clones that extended the
sequence another ~1 kb in the 5' direction were found. All the clones
were sequenced with multiple internal primers to generate the
full-length bTIMAP cDNA sequence.
Northern blots. Total RNA was harvested from various cells and from rat tissues using TRIzol (GIBCO Life Technology, Rockville, MD) and then subjected to extraction with phenol-chloroform. RNA (10-20 µg) was fractionated on a 1% formaldehyde agarose gel and transferred to a nylon membrane. 32P-labeled probes were made from various cDNA inserts (Rediprime kit, Amersham, Piscataway, NJ) and purified using a G-25 column (Amersham). The blots were hybridized at 62°C overnight in PreHyb buffer (Amersham) containing the different probes at 106 cpm/ml buffer. The Northern blots were visualized by exposure of the blot to a Kodak film or use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To control for loading, blots were reprobed with ribosomal protein L19 or L27a cDNA or with glyceraldehyde-3-phosphate dehydrogenase cDNA.
cDNA constructs and probes.
An hTIMAP cDNA encoding the predicted open reading frame (ORF) was
generated by RT-PCR from human aorta endothelial cell mRNA using the 5'
primer 5'-GGAATTCGATGGCCAGTCACGTGGA CCT-3' and the 3'
primer 5'-CGGTCGACTAGGAGATACGGCAACAGCCATG-3', in which
BamHI and SalI sites are underlined. TIMAP(C564S)
was constructed in the same way, except the 3' primer had a single
nucleotide replacement, 5'-CGGTCGACTAGGAGATACGGGAACAGCCATG-3',
which produced a serine instead of a cysteine in the CAAX box.
The PCR products were digested with BamHI and
SalI and ligated into pEGFP(C1) vector in
BamHI/XhoI sites. The cDNA products were
sequenced fully. The fusion proteins encoded by these cDNAs express
enhanced green fluorescent protein (EGFP) at the NH2
terminus of TIMAP or TIMAP(C564S). The RDA products for plasminogen
activator inhibitor-1 (PAI-1), ribosomal protein L19, ribosomal protein
L27a, fibronectin, integrin-, and laminin-
1 cloned
into pBluescript were used as probes.
Subcellular localization. GEN, MDCK, and COS-7 cells were plated on glass coverslips coated with type I collagen on the day before transfection. On the next day, transfection with pEGFP, pEGFP/TIMAP, or pEGFP/TIMAP(C654S) plasmids was carried out with LipofectAMINE 2000 for MDCK and COS-7 cells and by Lipofectin (GIBCO BRL) for GEN cells. After 24 h, the transfected cells were observed with an Aus Jena microscope using epifluorescence. Photomicrographs were taken with an Olympus camera with Kodak Elite 400 slide film. For confocal microscopy, the coverslips were mounted in mounting buffer (100 mM n-propyl gallate, 50% glycerol in PBS, pH 7.4), and the samples were viewed with a Nikon microphot-FXA or a laser scanning confocal system (model MRC 600, Bio-Rad Laboratories) coupled to a Zeiss Axiophot microscope through a ×100 oil-immersion objective. Images were processed using Photoshop software (Adobe Systems).
Immunhistochemistry and immunofluorescence studies. Polyclonal antibodies were prepared by Zymed Laboratories (South San Francisco, CA) by immunizing rabbits with peptides representing aa 383-401 and aa 511-532 of hTIMAP. These peptides are conserved between hTIMAP and bTIMAP and are not found in MYPT3, the closest relative of TIMAP (see Fig. 3A) or in other known or predicted protein sequences. The immunoglobulins were purified using peptide affinity columns prepared with the Sulfolink kit (Pierce, Rockford, IL). Each polyclonal anti-TIMAP IgG recognized full-length, but not NH2-terminal (aa 1-90), TIMAP expressed in bacteria (not shown). In endothelial cell and rat renal glomerular lysates, both antibodies recognize two predominant bands at 67 kDa (expected molecular mass for full-length TIMAP) and at 120 kDa (not shown). The identity of the second band is not known. It may represent a posttranslationally modified TIMAP isoform or an as yet unknown sequence in which both epitopes chosen for immunization are conserved.
TIMAP expression was examined in neonatal and adult rat tissues previously fixed in 10% buffered formalin (pH 7.4) and embedded in paraffin (19). Sections (5 µm) were prepared, deparaffinized with xylene, and rehydrated in a descending series of ethanols. Endogenous peroxidases were quenched twice with 0.3% hydrogen peroxide for 10 min at room temperature. The sections were then treated with 0.4% pepsin in 0.01 N HCl at 37°C for 30 min, washed twice with PBS, and blocked with 0.5% nonfat dry milk and 5% goat serum for 30 min at room temperature. The sections were incubated in the same solution with anti-TIMAP antibodies (1:100) at 4°C overnight, allowed to equilibrate to room temperature for 20 min, and washed twice with PBS. Sections were incubated with biotinylated goat anti-rabbit antibodies (Vectastain Elite ABC kit) for 30 min at 37°C and then with streptavidin-horseradish peroxidase (HRP; Vectastain Elite ABC kit) for 10 min at 37°C and visualized with diaminobenzidine. For dual-label immunofluorescence, the reaction was performed as described above, except HRP was visualized with the Cy3 tyramide (NEL704, New England Nuclear Life Science Products, Boston, MA). The HRP was then quenched, as described above, and the sections were incubated in the dark with mouse monoclonal anti-smooth muscle actin antibody (1:400; clone 1A4, Sigma) overnight at 4°C and then with goat anti-mouse HRP (1:1,000; New England Nuclear, Boston, MA). Visualization of the mouse monoclonal reaction product was achieved with tyramide fluorescein amplification (NEL701A, New England Nuclear). Immunofluorescence studies reflect data from five separate experiments using tissues from five different animals.Nucleotide sequence accession number. The nucleotide sequence accession numbers in the GenBank database are AF362910 and AF362909 for hTIMAP and bTIMAP cDNA, respectively.
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RESULTS |
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Identification of TIMAP, a novel TGF-1-responsive gene, in
endothelial cells.
To identify TGF-
1-responsive genes in endothelial cells, GEN cells
were cultured in the presence or absence of TGF-
1 (5 ng/ml) for
6 h, and mRNA was isolated and analyzed by RDA (Fig. 1A). TGF-
1-upregulated and -downregulated RDA products
were purified, subcloned, and sequenced.
TGF-1-induced TIMAP repression requires protein synthesis and
histone deacetylase.
In the presence of the RNA synthesis inhibitor actinomycin D, TIMAP
mRNA abundance declined (Fig.
2A). When cells were treated concurrently with TGF-
1 and actinomycin D, the rate of decline of
TIMAP mRNA abundance was similar to that observed with actinomycin D
alone and also with TGF-
1 alone (Fig. 2A). These data
suggest that the decline in TIMAP mRNA in response to TGF-
1 is not
due to enhanced degradation.
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TIMAP full-length cDNA and genomic structure. The full-length bTIMAP cDNA sequence is 6,299 bp long and encodes an ORF of 568 amino acids. The putative translation initiation codon was assigned on the basis that 1) there are no upstream ATGs, 2) a stop codon is present 11 codons upstream from the initiation codon, and 3) a Kozak consensus sequence (GCCatgG) (13-15) was found surrounding the initiation codon.
The full-length hTIMAP cDNA was obtained by first establishing that KIAA0823 (35) (accession no. AB020630, GenBank), originally isolated from human brain, represents a partial human cDNA homologous to the bTIMAP cDNA. The full-length cDNA of hTIMAP is 6,113 bp long and encodes a 567-aa polypeptide. The amino acid sequence of hTIMAP is 97% identical to that of bTIMAP. The predicted TIMAP protein contains strong 5' nuclear localization consensus sequences, 5 ankyrin repeats within its NH2-terminal half, a protein phosphatase-1 (PP1) binding domain just upstream of the first ankyrin domain, and a CAAX box (CRIS) at the COOH terminus (Fig. 3B).
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TIMAP mRNA expression.
All cultured endothelial cells (bovine GEN and BAE cells and human
aorta, umbilical vein, and microvascular endothelial cells) examined so
far express TIMAP mRNA. TIMAP transcript was also observed in several
hematopoietic cell lines (KG-1a, Molt4, Dami, and TF1; Fig.
4A). However, TIMAP was not
detected in HeLa cells, WM793 melanoma cells, and HCT116 colorectal
cells (Fig. 4A). In HEK-293 cells, a human embryonic kidney
cell line, two transcripts larger than the TIMAP mRNA observed in other
cells hybridized with the TIMAP cDNA probe. Whether these mRNAs are
derived from the TIMAP gene, with alternate transcription start sites,
or represent homologous mRNA not derived from the TIMAP gene remains to
be determined.
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In vivo, TIMAP is expressed predominantly in endothelial cells.
Two distinct polyclonal anti-TIMAP peptide antibodies resulted in
essentially identical staining patterns in vivo. In newborn kidney, the
TIMAP antibodies localized predominantly to the vasculature, with
strong staining of endothelial cells in arteries and arterioles (Fig.
5, A and B).
Dual-immunofluorescence studies demonstrated that staining was excluded
from -smooth muscle actin-positive cells (Fig. 5, C and
D). Specific staining of the renal vasculature without
staining of renal parenchymal epithelial cells is shown in Fig.
5D. In adult kidney and heart, TIMAP antibodies similarly localized to blood vessels (not shown).
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TIMAP is a novel CAAX box protein localized at the plasma membrane.
TIMAP contains a CAAX box at the COOH terminus and also contains
another cysteine within 12 amino acids of the CAAX box. This suggests
that TIMAP is a novel CAAX box protein subject to prenylation and
plasma membrane localization. Two mammalian expression vectors were
prepared: one encoding a green fluorescent protein (GFP)-TIMAP fusion
protein and the second a GFP-TIMAP(C564S) fusion protein in which the
cysteine in the CAAX box was replaced by a serine by site-directed
mutagenesis. This mutation has previously been shown to abolish
membrane association of CAAX box proteins (8, 12, 25). The
GFP-TIMAP fusion proteins were then expressed transiently in GEN,
COS-7, and MDCK cells. Data for MDCK and GEN cells are shown in Fig. 5.
Identical results were obtained from experiments with COS-7 cells (not
shown). At 24 h after transfection, control GFP not fused to TIMAP
was diffusely expressed in all cell types, tending to localize most
strongly to the nucleus (Fig. 6,
A and D). The GFP-TIMAP fusion protein was
expressed at the cell membrane and in the perinuclear region (Fig. 6,
B and E). By contrast, the GFP-TIMAP(C564S)
fusion protein did not localize to the plasma membrane (Fig. 6,
C and F). These results suggest that, similar to
other CAAX box proteins, TIMAP localizes to the cell membrane
and that membrane localization requires posttranslational prenylation
at the COOH terminus. In MDCK (Fig. 6F) and COS-7 (not
shown) cells, TIMAP(C564S) was also found in the nucleus, consistent
with the NH2-terminal nuclear localization signal.
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DISCUSSION |
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This study describes the initial characterization of a novel gene
and its protein product TIMAP, also referred to as PPP1R16B. The TIMAP gene, located on human chromosome 20q11.22-12, contains 10 exons and spans >87 kb. Its mRNA is highly expressed in cultured endothelial and hematopoietic cells and in the CNS, adrenal, lung, and
spleen in vivo. The TIMAP protein is highly expressed in vascular endothelial cells in vivo. Steady-state TIMAP mRNA levels in
endothelial cells decrease significantly in response to TGF-1, an
effect that appears to be due to a protein synthesis- and
HDAC-dependent reduction in transcription. The predicted TIMAP protein
contains strong NH2-terminal nuclear localization signals,
a PP1-interacting domain, five ankyrin repeats in its
NH2-terminal half, and a COOH-terminal CAAX box motif. When
expressed transiently in endothelial and epithelial cells, the
wild-type TIMAP protein localizes to the plasma membrane, and a point
mutation in the CAAX box, predicted to eliminate COOH-terminal
prenylation, abolishes membrane localization. Hence, TIMAP is a novel,
TGF-
1-regulated CAAX box protein highly expressed in endothelial and
hematopoietic cells in vitro and in vascular endothelium and the CNS in vivo.
The TIMAP cDNA sequence was found by RDA screening of glomerular
capillary endothelial cells for unknown transcripts subject to
regulation by TGF-1. The RDA product TIMAP was further
characterized, because a previously identified homolog, the 5,597-bp
partial human cDNA KIAA0823 (35) (accession no.
AB020630, GenBank) predicted a COOH-terminal CAAX box,
suggesting that TIMAP could represent a novel signaling molecule. The
predicted ORFs for hTIMAP and bTIMAP are 1,701 and 1,704 bp long,
respectively, and both cDNAs contain long (4,411 and 4,407 bp,
respectively) 3'-untranslated regions. The predicted proteins are 568 (bovine) and 567 (human) amino acids long, and the amino acid sequences
are highly conserved (97% homologous) between the two species. Three
overlapping human genomic clones that map to chromosome 20q11.22-12
were found which contain the entire TIMAP cDNA as 10 exons. The total
genomic span of the TIMAP sequence is just over 87 kb. Whether
alternate upstream exons could contribute to larger transcripts, such
as those observed in HEK-293 cells (Fig. 4A), is unknown.
One conserved motif found in the predicted TIMAP protein sequence is a CAAX box (CRIS) at its COOH terminus. The CAAX motif, always located at the COOH terminus, predicts posttranslational modification through prenylation, which then tends to target the protein to the inner leaflet of the plasma membrane (8). The prenylation of CAAX box proteins renders their COOH terminus highly hydrophobic and, thus, capable of interacting with the membrane phospholipid bilayers. The finding that the terminal amino acid of the putative TIMAP protein is serine would predict that it is a substrate for a farnesyltransferase (8).
The CAAX motif is found in a restricted set of protein families, among
them Ras family members, nuclear lamins, and the -subunit of
trimeric G proteins (8). Mutations of the cysteine
residue, to prevent prenylation, interfere with membrane localization
of CAAX box proteins and, in some cases, also with other
protein-protein interactions (12). In the case of TIMAP,
the wild-type protein localized to the plasma membrane of endothelial
and epithelial cells when transiently expressed (Fig. 6). The C564S
point mutation in which cysteine is replaced with serine in the
CAAX box of TIMAP resulted in loss of this membrane localization and
retention of the protein in large cytoplasmic inclusions (Fig. 6,
c and f). Hence, the functional
significance of the CAAX box, at least for membrane localization of
TIMAP, has been established. In COS-7 and MDCK cells, but not
in endothelial cells, transiently expressed GFP-TIMAP(C564S)
fusion protein was found in the nucleus. It is likely that nuclear
localization is mediated by the nuclear localization sequences found in
the TIMAP NH2 terminus (Fig. 3B). The difference in the ability of TIMAP(C564S) to localize to the nucleus in epithelial vs. endothelial cells is not understood.
The ORF of the TIMAP sequence also predicts five ankyrin repeats in its NH2-terminal half. Ankyrin repeats generally mediate protein-protein interactions. There is a large diversity of ankyrin repeat-containing proteins with many different intracellular locations and functions (31). To our knowledge, no other family of proteins containing ankyrin repeats and CAAX box has been described.
The TIMAP protein shares significant domain homology with a recently cloned protein named MYPT3. At the amino acid level, there is 44.7% homology between TIMAP and MYPT3. The MYPT3 protein was discovered by the yeast two-hybrid approach using PP1 as the bait (32). MYPT3 was shown to bind PP1 and to have inhibitory activity toward this phosphatase. MYPT3 functions are similar to those of two other proteins, MYPT1 and MYPT2, which share the ankyrin and PP1 binding domains with MYPT3 and TIMAP (Fig. 3B). However, because the MYPT1 and MYPT2 proteins do not share the CAAX box found in the PPP1R16B family of proteins, they are more distantly related to TIMAP than is MYPT3 (Fig. 3C).
The TIMAP transcript abundance in endothelial cells declines within
2-4 h in after addition of TGF-1 to the cells (Figs. 1D and 2A). When new RNA synthesis was blocked
with actinomycin D, the TIMAP mRNA level declined at a rate very
similar to that observed in cells treated with TGF-
1 alone or with a
combination of TGF-
1 and actinomycin D (Fig. 2A). These
findings suggest that the effect of TGF-
1 on TIMAP mRNA abundance
does not reflect a change in TIMAP mRNA stability. Inasmuch as TGF-
1
did not alter TIMAP transcript levels in the presence of CHX, it is
evident that TGF-
1-mediated regulation of TIMAP mRNA levels is
indirect and requires new protein synthesis. TGF-
1-dependent
transcriptional repression involving proteins that assemble in
multimeric complexes with HDAC is now well described. The proteins Ski,
SnoN, and TGIF can associate with activated Smad complexes, resulting
in recruitment of corepressors and HDAC to Smad-responsive promoters
(34, 40, 41). In this study, TGF-
1-mediated reduction
in TIMAP transcript abundance was sensitive to TSA, a potent inhibitor
of HDAC. The dependence of TIMAP mRNA downregulation on HDAC could
reflect transcriptional repression of TIMAP.
We observed abundant expression of TIMAP in cultured bovine and human
endothelial cells derived from large and small blood vessels. The TIMAP
SAGE tag was found in the two public human endothelial cell SAGE
libraries and in a normal colon endothelial cell library
(33) at a frequency >30-fold higher than its expression frequency in all public nonendothelial libraries (~4 × 106 tags) combined. These findings suggested that TIMAP is
highly expressed in endothelial cells. Two distinct polyclonal peptide antibodies representing TIMAP epitopes not found in MYPT3 or other known or predicted protein sequences localized predominantly to the
vasculature. In blood vessels examined by dual-label immunofluorescence with TIMAP and -smooth muscle actin antibodies, TIMAP was excluded from the smooth muscle cell layer, confirming the endothelial cell
location. Hence, SAGE expression surveys, Northern blot analysis of
cultured cells, and histology confirm that TIMAP is expressed in
endothelial cells.
We also observed TIMAP mRNA expression in a number of transformed hematopoietic cell lines. There are no reported SAGE tag libraries for hematopoietic cell lines; hence, no further information on the level of TIMAP expression in cells of that lineage could be obtained from that source. Because endothelial and hematopoietic cells are derived from common precursors, it is tempting to speculate that TIMAP may be functionally important in cells derived from the hemangioblast lineage.
We did observe expression of TIMAP mRNA in several rat tissues, including all portions of the CNS examined. In this regard, it is also of note that the KIAA0823 cDNA was initially cloned from a human brain cDNA library (35). Furthermore, bulk medulloblastoma and astrocytoma and bulk thalamus SAGE libraries contained the TIMAP tag. It therefore seems likely that TIMAP is also expressed in the CNS. Clearly, more work will need to be done to further define the localization of TIMAP expression in vivo.
In conclusion, we describe the cDNA and putative amino acid sequence of
a novel CAAX box protein TIMAP, the CAAX box mediating localization of
transiently expressed protein, to the plasma membrane. The TIMAP
protein is predicted to contain NH2-terminal nuclear localization signals, multiple ankyrin repeats, and a PP1 binding domain. To our knowledge, the PPP1R16B family of proteins, of which
TIMAP and MYPT3 are members, is the first to contain the combination of
multiple ankyrin repeats and CAAX motif. The TIMAP mRNA is highly
expressed in endothelial and hematopoietic cells in culture and in
vascular endothelium in vivo. We postulate that transcriptional
repression of TIMAP may be an important component of the apoptotic
and/or capillary morphogenesis response of endothelial cells to
TGF-1.
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ACKNOWLEDGEMENTS |
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The electronic SAGE libraries for normal and tumor endothelium from colon were kindly provided by B. Vogelstein. V. Senchak provided expert cell culture assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-50764 (B. J. Ballermann) and HL-56091 (B. J. Ballermann and C. J. Lowenstein). W. Cao was supported by the Maryland Chapter of the National Kidney Foundation.
Address for reprint requests and other correspondence: B. J. Ballermann, Albert Einstein College of Medicine, Ullmann Bldg. 619, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: bjballer{at}aecom.yu.edu).
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.
First published February 27, 2002;10.1152/ajpcell.00442.2001
Received 14 September 2001; accepted in final form 18 February 2002.
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