Trihydrophobin 1 is a new negative regulator of A-Raf kinase.

Our previous work indicated that instead of binding to B-Raf or C-Raf, trihydrophobin 1 (TH1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1-372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase-dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G(0)/G(1) phase-arrested. Taken together, our data provide a clue to understanding the cellular function of TH1 on Raf isoform-specific regulation.

The human trihydrophobin 1 (TH1) 1 gene is the homolog of Drosophila TH1, which was originally identified during the positional cloning of mei-41 (1,2). The TH1 gene lies adjacent to mei-41 and was characterized further by the D. T. Bonthron group in 2000 (3). According to their studies, the TH1 gene was highly conserved from Drosophila to human by sequence comparison. The human TH1 gene was located in chromosome 20q13, which had a transcript product of 2.4 kb. Multiple tissues detected by Northern blots showed that TH1 was widely expressed. The human TH1 protein has been predicted having a molecular mass of 65.8 kDa and displays high levels of expression in cardiac and skeletal muscle, kidney, adrenal, and thyroid. Although ubiquitously expressed, its function is not clear at present. From protein sequence data-base analysis, this protein does not seem to have a potential kinase domain. Our previous work has identified TH1 as a new interaction partner of A-Raf kinase, which belongs to a serine/threonine kinase family involved in MAPK signal transduction pathways (4).
The mammalian MAPK signaling pathways have been found to play key roles in a wide range of cellular responses such as proliferation, differentiation, and apoptosis corresponding to multiple extracellular stimuli (5)(6)(7)(8)(9). Among them, the Raf/ MEK/ERK pathway is so far the best described pathway, which is responsive to mitogenic and differential stimuli. It was depicted as a conserved three-kinase cascade pathway that consists of MKKKs, MKKs, and ERKs (6 -9). As members of MKKKs, the serine/threonine Raf kinase family is poised in the core of the three-kinase module, which is important in relaying upstream signals to the nucleus (10 -12). In mammalian cells, the Raf kinase family is composed of three important isoforms: A-Raf, B-Raf, and C-Raf (also known as Raf-1). These Raf isoforms share high similarities in their sequence and structure as reported previously (11)(12)(13).
Most studies have revealed that the Raf isoforms are under different regulation and have individual functions. In brief, as to their activation, although all three Raf isoforms interact with their upstream activator Ras and are activated by translocating to the membrane (14,15), studies in COS-7 cells showed that their activations were quite different. Unlike B-Raf, whose activation is sufficient by oncogenic Ras alone, A-Raf behaves like C-Raf, which needs synergism of oncogenic Ras and other tyrosine kinases such as Src to achieve full activation (16,17). Adding to the complexity, B-Raf kinase activity was proven to be determined by Ras and other members of the small G protein family such as Rap1 in PC12 cells (18) or TC21 in NIH3T3 cells (16,19,20). Moreover, Rap1 has been reported to inhibit C-Raf, whereas TC21 interacts with B-Raf and C-Raf but not A-Raf in these studies. Upon activation, the three Raf isoenzymes display differences in their catalytic activities. As measured by protein kinase assays, B-Raf has the highest activity to phosphorylate their common substrate, MEK1, whereas A-Raf has the lowest activity, and C-Raf catalytic activity is intermediate between them (21,22). Studies with dominant negative mutants (23) and antisense oligodeoxynucleotides (24 -27) on Raf isoforms suggested that Raf family activities are also regulated differentially by multiple pathways including phosphatidylinositol 3-kinase (23) 1 The abbreviations used are: TH1, trihydrophobin 1; ERK, extracellular signal-regulated kinase; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorter; GST, glutathione S-transferase; HA, influenza hemagglutinin monoclonal antibody epitope; HEK, human embryonic kidney; KD, kinase-dead; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase; MKK, mitogen-activated protein kinase kinase; MKKK, mitogen-activated protein kinase kinase kinase; MOPS, 4-morpholinepropanesulfonic acid. stimulus. For instance, cAMP-dependent protein kinase A can activate Ras-related protein Rap1 (18,30) and selectively activates B-Raf but inhibits C-Raf (31)(32)(33). Besides, A-Raf is regulated distinctly from the other two Raf isoforms in the interleukin-3-induced phosphatidylinositol 3-kinase signal pathway (23). Although some common functions are found in the Raf family, their functions are noncompensable (13, 14, 21, 34 -36). As reported, their distribution patterns are quite different. In contrast to C-Raf protein, which is ubiquitously expressed, the expression of A-Raf has a tissue-specific distribution and is expressed predominantly in urogenital tissues, whereas B-Raf is found mostly in brain tissues (37,38). The restricted expression pattern may suggest that A-Raf has unique functions in such tissues. With gene knockout technology, mice deleted of different Raf genes show very different phenotypes (39 -41). In previous studies, Sewing et al. (42) reported that high intensity of Raf activity caused cell cycle arrest mediated by p21 Cip1 in mouse embryo fibroblasts. Recent studies in NIH3T3 murine fibroblast cells have shown that only moderate Raf activation such as A-Raf and C-Raf was necessary for G 1 progression and cell proliferation, whereas the strongest activation of B-Raf led to apoptosis (43). Thus, as a member of the Raf family, A-Raf may have its specific regulator and play a unique role in cells.
In contrast to C-Raf and B-Raf, as increasingly demonstrated in recent studies, A-Raf has its unique interaction proteins. Besides binding to Ras (14, 44 -53), MEK (35,42,45,50,53), and 14-3-3 (54 -56), A-Raf has been shown to interact with casein kinase 2␤ (36,57) and pyruvate kinase type M2. Casein kinase 2␤ has been proven to be a specific activator of A-Raf which enhanced A-Raf kinase activity in Sf9 cells (36). Pyruvate kinase type M2 is a pyruvate kinase that correlates A-Raf with energy metabolism in tumorigenesis (13). Yuryev et al. (58) also discovered that two new proteins, hTOM and hTIM, which are involved in the mitochondrial transport system, interact with A-Raf, located exclusively in mitochondria (58). Our recent study also reported that TH1 can interact directly with A-Raf, but how this interaction mediated A-Raf function has not been determined.
Here, we observed by confocal microscopy that TH1 colocalizes with A-Raf in SMMC-7721 hepatocarcinoma cells and COS-1 cells. By mapping the A-Raf binding region in TH1, the N-terminal 1-372 amino acids are found sufficient for binding with A-Raf. We also examined the association between TH1 and A-Raf in a different cellular context and found that the activation of A-Raf could enhance its association with TH1. In combination with the inhibitory effect of TH1 on A-Raf kinase tested by protein kinase assays, we demonstrated that TH1 is a new specific suppressor of A-Raf kinase in the MAPK/ERK cascade and the inhibition of cell cycle progression by TH1 may proceed through its interaction with A-Raf kinase.

EXPERIMENTAL PROCEDURES
Cell Lines and Reagents-The human SMMC-7721 hepatocarcinoma cells (Institute of Cell Biology, Academic Sinica) were maintained in RPMI 1640 supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 50 g/ml streptomycin at 37°C under 5% CO 2 in humidified air. The human embryonic kidney 293 (HEK293) cells kindly provided by the National Key Laboratory of Neurobiology of Fudan University and African green monkey kidney COS-1 cells (Institute of Cell Biology, Academic Sinica) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 50 g/ml streptomycin. TH1 fragment F5 and the full-length TH1 stable transfectants derived from SMMC-7721 cells were established by selection in the culture medium with 4 mg/ml G418 (Invitrogen) using LipofectAMINE (Invitrogen). Leupeptin, aprotinin, and phenylmethylsulfonyl fluoride were purchased from Sigma.
Yeast Interaction Assays-For interaction in yeast, a series of TH1 deletion mutants constructed into plasmid pLexA were cotransformed with pB42AD-A-Raf and empty pB42AD plasmid to verify the specificity of the two-hybrid assay using EGY48 yeast strain (Mat␣ trp1 ura3-52 leu2::pLeu2-lexAop6(⌬G-UAS leu2)). Yeast cotransformation was performed by the lithium acetate method provided by the manufacturer (Clontech).
In Vitro Binding Assay-GST-TH1, its GST-fused deletion mutants, and A-Raf protein prepared as pcDNA3 constructs were used to generate [ 35 S]methionine-labeled proteins individually with the TNT® Coupled Reticulocyte Lysate System (Promega) according to the manufacturer's instructions. A GST pull-down assay was performed as described previously (4, 59, 60). In brief, the labeled proteins were addressed immediately by constant mixing with 25 l of glutathione-Sepharose beads in the binding buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol, 10 mM NaF, 1% Nonidet P-40, 1 mM NaVO 4 , 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) for 3 h at 4°C. Then the beads were washed three times with the binding buffer and boiled in SDS sample buffer. The bound proteins were analyzed by autoradiography after they were resolved by 12% SDS-PAGE.
In Vivo Binding Assay-The COS-1 and HEK293 cells in 60-mm dishes (Nunc) were transiently transfected with 3 g of pcDNA3.1myc-TH1 plasmid alone or with the combination of 0.5 g of pcDNA3-Ras and 0.5 g of pcDNA3-Src. Total DNA was kept constant by pcDNA3.1-myc-his empty vector. 48 h after transfection, the cells were starved in 0.1% fetal bovine serum medium for 18 h and then stimulated by the readdition of serum. Cell lysates were prepared, and immunoprecipitation was performed as described previously (4,59,60). In brief, ϳ500 g of cell lysates was incubated with mouse normal IgG or 2 g of anti-myc monoclonal antibody at 4°C for 2 h. The preequilibrated protein G-agarose beads were then added. After 2 h of incubation, the precipitates were collected by centrifugation and washed gently three times with the lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl 2 , 25 mM ␤-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The bound proteins were eluted by boiling in SDS sample buffer and resolved on a 10% SDS-polyacrylamide gel. The proteins were analyzed by immunoblotting with a 1:200 dilution of a polyclonal anti-A-Raf antibody or with a 1:5,000 dilution of monoclonal anti-myc antibody, respectively. Proteins were detected using the ECL kit.
In Vitro Raf Kinase Assays-For each Raf isoform kinase assay, COS-1 cells or HEK293 cells were collected by centrifugation and washed twice with phosphate-buffered saline before being lysed in lysis buffer as described previously (59,60). 500 g of cell lysates was incubated with 2 g of anti-A-Raf antibody or anti-B-Raf or anti-C-Raf antibody for 3 h and then mixed constantly with protein G-agarose beads at 4°C overnight. The immunoprecipitates were analyzed for kinase activity using a Raf-1 kinase cascade assay kit (Upstate Biotechnology). Briefly, 0.4 g of inactive GST-MEK1 and 1 g of inactive GST-ERK1 were incubated with each precipitated Raf in 20 l of kinase buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) containing either 10 Ci of [␥-32 P]ATP or nonradioactive ATP for 30 min at 30°C. Then, myelin basic proteins were used as activated ERK substrates by incubation for 10 min. GST-MEK1 phosphorylation was analyzed by 10% SDS-PAGE and autoradiography. Alternatively, the phosphorylation of GST-MEK1 was detected by anti-p-MEK1 antibody. The quantitation of MEK1 phosphorylation by Raf kinases was determined by phosphorimaging or by liquid scintillation counting. Alternatively, most studies have employed prokaryotic expressed MEK1/MEK1 (K52) or ERK1 as substrates in the kinase assay. We also prepared such purified protein in repeating this experiment as reported previously (4,25).
FACS Cell Analysis and Cell Count-1-3 ϫ 10 6 7721 cells, pcDNA3-TH1 F5, and pcDNA-3.1-myc-TH1 stably transfected cells were cultured in RPMI 1640 medium for 48 h before collection by centrifugation. Cells were washed and fixed with 70% ethanol for the FACS cell cycle analysis as described previously (4, 59 -61). After fixation in ethanol for 12 h, cells were washed again with phosphatebuffered saline containing 1% bovine serum albumin and then stained with a solution containing 50 g/ml propidium iodide, 250 g/ml RNase, and 0.1% Triton X-100 and analyzed by quantitative flow cell cytometry with standard optics of FACScan flow cytometer (BD PharMingen FAC-Star) employing the Cell Quest program.

Confocal Microscopic Analysis of TH1 and Raf Family-In
previous work (4) we reported that TH1 can bind specifically to A-Raf both in vitro and in vivo. To elucidate the subcellular localization of TH1 with different Raf isoenzymes, we selected COS-1 cells to transfect with pEGFP, pEGFP-TH1, and pD-sRed-A-Raf, -B-Raf, and -C-Raf plasmids, respectively. 48 h after transfection, the cells were fixed and analyzed under confocal microscopy. As shown in Fig. 1a, the EGFP is distributed in whole cellular compartments, especially in the nucleus, whereas red fluorescent protein-fused A-Raf is distributed exclusively in the cytoplasm. There is no colocalization between them (Fig. 1a). The EGFP-fused TH1 protein is also distributed in both the cytoplasm and nucleus (Fig. 1, c and d). After coexpressing EGFP-fused TH1 and red fluorescent protein fused A-Raf proteins, we detected the cells with fluorescence microscope. By merging the separate projection images greenonly and red-only emission detection, we observed that the double-transfected cells contained yellow granules indicating colocalization of TH1 and A-Raf (Fig. 1b). As a further support to our former results, the other two members of Raf family, B-Raf and C-Raf, did not colocalize with TH1 when checked by confocal microscopy (Fig. 1, c and d). We also got the same results using SMMC-7721 hepatocarcinoma cells. Taken together, they confirmed the specific interaction between TH1 and A-Raf in vivo.
Mapping of A-Raf Binding Regions in TH1-To investigate the region in TH1 responsible for binding A-Raf, we first constructed a series of TH1 deletion mutants into plasmid pLexA and tested for their binding abilities with A-Raf in a yeast system. These mutant constructs were cotransformed either with the empty pB42AD plasmid or with pB42AD-A-Raf into EGY48 yeast cells. Cotransformants were tested for growth in the absence of leucine and production of ␤-galactosidase. As shown in Fig. 2A, other than cotransformants containing fulllength of TH1, the interaction between C-terminal truncated mutants TH1 F1 (amino acids 1-452) and A-Raf could also be detected through nutrition and color selection. Further deletion of TH1 F1 (1-452) still showed an interaction when amino acids 1-372 were present. This interaction was disrupted when TH1 F2 (amino acids 1-372) was further deleted as TH1 F3 (amino acids 1-192) and TH1 F4 (1-170). Interestingly, the N-terminal 164 amino acids truncated mutant TH1 F5 (amino acids 164 -452) lost the ability to bind with A-Raf, suggesting that the N-terminal of TH1 was important for binding with A-Raf. We also found that no other TH1 deletion mutants had the ability to bind with A-Raf in yeast interaction assays. To confirm further the interaction, a GST pull-down experiment was performed by expressing GST-fused TH1, GST-fused TH1 deletion mutants, and A-Raf protein, respectively. As shown in Fig. 2B, A-Raf could bind directly with TH1 F1 (amino acids 1-452) and TH1 F2 (amino acids 1-372), whereas no binding with TH1 F5 (164 -452) was detected. Taken together, the N-terminal 1-372 amino acids of TH1 were necessary and sufficient for the interaction with A-Raf.
Enhanced Binding of TH1 with Activated A-Raf-Studies have shown that exposure of cultured cells to diverse mitogens, including serum, growth factor, and phorbol ester, results in activating a certain cascade of protein kinases and transduction signals from the membrane into the nucleus (62,63). A-Raf acts as a key intermediate kinase in the cascade of the MAPK/ ERK pathway whose kinase activity was alternatively activated or deactivated depending on cellular context. To clarify whether the A-Raf kinase activity state affects its association with TH1, we tested their interaction upon different stimuli. Because serum contains various components including epidermal growth factor and platelet-derived growth factor and complement body, which had been reported to activate the MAPK/ ERK pathway in former studies, we applied it as a stimulus. Other stimuli used were A-Raf upstream activator and constitutive activated oncogene Ras/Src, which are regarded as strong stimuli that lead to the greatest Raf protein kinase activity when expressed in combination (14,16,62). For binding assays, COS-1 cells transfected with different combinations of plasmids were serum starved for 18 h and then retreated with the addition of serum or untreated. As shown in Fig. 3A,  3,4,5). This suggests that TH1 and A-Raf may exist in a protein complex in cells and that complex formation does not require growth factor stimulation. We also found that strengthened bindings between activated A-Raf and TH1 were observed. The increased binding of A-Raf kinase to TH1 was correlated with the extent of its enzymatic activities, whereas the levels of total A-Raf protein were not changed (Fig. 3A, middle panel). As a control, the levels of TH1 protein precipitates were determined by a monoclonal antibody against the myc epitope (Fig. 3A, bottom panel). A similar result was achieved when we used HEK293 cells, which expressed a high level of endogenous A-Raf protein. As shown in Fig. 3B, more evidently, with serum and/or oncogenic Ras/Src stimulation, TH1 binds increasingly with samples containing activated A-Raf (lanes 4 -6) rather than unstimulated sample (lane 3). To verify further whether A-Raf kinase activity plays a pivotal role in its association with TH1, we used a eukaryotic expression plasmid pBOS-A-Raf(KD)-(HA) 2 (23) to express ki-nase-dead A-Raf protein to test its interaction with TH1. As shown in Fig. 3C, kinase-dead A-Raf protein still could bind to TH1 but showed no quantity difference in binding with TH1 upon stimulation. Direct comparison of the bindings between wild type A-Raf and kinase-dead A-Raf with TH1 reveals that TH1 preferentially associates with wild type A-Raf (Fig. 3D). Given these, it might be concluded that A-Raf activation state is essential for its association with TH1 in both cell lines. The increasing ability of activated A-Raf to bind with TH1 suggests that TH1 may play some role in regulating A-Raf kinase.
Overexpression of TH1 Inhibits A-Raf Kinase Activity upon Serum Stimulation-A-Raf is well known as an upstream activator of MEK1, which regulates MEK1 kinase activity in the MAPK/ERK pathway (13,53). To investigate the relevance between the interaction and Raf kinase activities, we performed Raf protein kinase assays using kinase-deficient GST-MEK1 as substrate. Before starting the experiments, we tested the cross-reactivity of the A-, B-, and C-Raf antibodies purchased, respectively. We found that certain Raf antibodies failed to immunoprecipitate the other two Raf proteins (data not shown). COS-1 cells were transfected with different amounts of plasmids pcDNA3.1-myc-TH1 and were serum starved for 18 h before restimulation by complete medium containing 10% serum. The whole cell lysates from transfected cells and mock cells were immunoprecipitated with an anti-A-Raf polyclonal antibody. The in vitro kinase assay of the A-Raf immunoprecipitates revealed that A-Raf kinase activity was decreased significantly in the presence of TH1 in a dose-dependent manner (Fig. 4A). As a control, the A-Raf bindingdeficient TH1 fragment F5 was introduced and had no effect on A-Raf kinase activity as shown in Fig. 4A (lane 1). It reflects that the association between TH1 and A-Raf is important for TH1 to regulate A-Raf kinase activity. We also performed the same kinase assay using HEK293 cells and anti-p-MEK1 antibody instead of using 32 P incorporation. The data confirmed the result in COS-1 cells (Fig. 4A, right panels). Because in our previous work, TH1 could interact specifically with A-Raf but not B-/C-Raf, we checked the other two Raf isoforms kinase activities under the same experimental conditions. As shown in Fig. 4B, neither B-Raf nor C-Raf kinase activity was influenced by TH1 overexpression. Liquid scintillation detection of phosphorylation of myelin basic protein substrates depicted the different effects of TH1 overexpression on each Raf kinase (Fig.  4C). It can be inferred that through direct specific interaction with A-Raf, TH1 uniquely suppressed A-Raf kinase activity but not B-/C-Raf kinase activities.
Overexpression of TH1 Inhibits the Kinase Activity of A-Raf Activated by Oncogenic Ras/Src-Although Ras is the best characterized upstream activator of Raf isoforms, it still needs other components including tyrosine kinases to achieve full activation of Raf kinases (16). We investigated further the effect of TH1 overexpression on A-Raf kinase activity upon constitutively activated oncogenic Ras (Val 12 ) and Src (Y527F) stimulation. As shown in Fig. 5, COS-1 cells were transiently transfected with Ras (lanes 3 and 4) or Src (lanes 5 and 6) individually or in combination (lanes 7 and 8). When transiently expressing TH1 as indicated in Fig. 5 (lanes 4, 6, and 8), endogenous A-Raf precipitated by anti-A-Raf antibody was tested for its ability to phosphorylate GST-MEK1 in an in vitro kinase assay. Our data suggested that TH1 suppressed A-Raf kinase activities stimulated by Ras and/or Src. Combined together, TH1 functions as a negative regulator of A-Raf in the Ras-mediated MAPK/ERK pathway.
TH1 Overexpression Influenced SMMC-7721 Cell Cycle Progression and Cell Proliferation-Raf proteins have been implicated in regulating cell cycle progression or cell cycle arrest depending on their effects on both ERK signal intensity and duration. Studies have also indicated that A-Raf played an important role in cell cycle progression and proliferation in FIG. 2. Mapping of the regions responsible for A-Raf binding in TH1. A, schematic representation of the interaction between A-Raf and TH1 deletion mutants with the yeast two-hybrid system. Constructs were engineered as a fusion protein with the DNA binding domain LexA or with GST as described under "Experimental Procedures." Regions and residues numbers are indicated. The column on the right summarizes whether constructs did (ϩ) or did not (Ϫ) interact. B, in vitro interaction between selected GST-TH1 and GST fused TH1 deletion mutants with A-Raf. TH1, TH1 deletion mutants, and A-Raf protein were [ 35 S]methionine labeled and used for interaction. Molecular mass markers are indicated on the left, and individual proteins are indicated on the right. vascular smooth muscle cells (24) and in hemopoietic cells (23). Because TH1 could inhibit A-Raf kinase activity, we wished to check its effect on cell cycle progression. We established several cell strains that stably express TH1 (TH1/7721) and GST-TH1 F5 (F5/7721) in SMMC-7721 hepatocarcinoma cells and COS-1 cells. With flow cell cytometry analysis, cells stably expressing TH1 were found to be G 0 /G 1 phase-arrested and cells in S phase and G 2 /M phase were greatly diminished compared with 7721 cells and pcDNA3/7721 cells (Fig. 6A). However, the cells that express A-Raf-binding-deficient TH1 mutant F5 could not affect the cell cycle progression. At least three independent experiments were performed, and statistical comparison of the changes of cell cycle was presented in Fig. 6B. From growth curves (Fig. 6C), we could see that cells stably expressing TH1 grew much more slowly than mock cells, whereas TH1 F5 lack this ability, which was consistent with the FACS results. Taken together, TH1 overexpression does affect the biological behavior of SMMC-7721 hepatocarcinoma cells and COS-1 cells.

DISCUSSION
In mammalian cells, A-Raf belongs to the Raf kinase family, which consists of other two members, B-Raf and C-Raf, acting as a key intermediate that relays signals from upstream Ras and tyrosine kinases to downstream serine/threonine kinases. Sequence comparisons of all three Raf proteins reveal that they share a high degree of similarity, consisting of three conserved regions, CR1, CR2, and CR3. Among them, CR1 and CR2 form the N-terminal regulatory domain, and CR3 comprises the catalytic domain. The N-terminal regulatory domain functions to suppress the catalytic activity of Raf, and removal of this domain will lead to the constitutively active forms of all three Raf kinases. The C-terminal catalytic domain is mainly responsible for phosphorylation of downstream substrates (13, 64 -66). The alignment of the three Raf isoforms indicates that the C-terminal kinase domain is highly conserved, and the Nterminal regulatory domain is less conserved. The variety of the N-terminal regulatory domain may account for the specific regulation of each Raf isoform. Our previous work has identified TH1 as a specific A-Raf interaction protein that is uniquely bound to the N-terminal 1-162 amino acids of A-Raf. These amino acid sequences correspond to the A-Raf CR1 regulatory domain, which contains the Ras-binding domain and the cysteine-rich domain responsible for Ras binding and A-Raf activating. Besides Ras, other molecules such as 14-3-3 and a putative lipid ligand were reported to bind to the cysteine-rich domain regardless of different Raf isoforms (10). TH1 may be unique in regulating A-Raf rather than B-Raf or C-Raf through specific binding with this domain.
The biological reasons underlying Raf diversity are not fully understood but imply that Raf isoforms have overlapping and unique functions. The regulation of Raf isoforms was a complex process in which different Raf isoforms combine common and unique mechanisms regulate Raf kinase activity as reported previously. Studying the molecular mechanism regulating each specific reaction has only begun in recent years. A specific reaction may be regulated by a specific protein-protein interaction. Studies on Raf isoforms have revealed that each Raf protein has specific interaction partners other than common activators and effectors. For instance, PA28␣, a subunit of the 11 S regulator of proteosomes, was found to bind specifically with B-Raf but not A-Raf or C-Raf. Meanwhile, A-Raf also has its unique interaction proteins such a casein kinase 2␤, pyruvate kinase type M2, hTOM, and hTIM. Our previous study on A-Raf also depicted TH1 as a new specific interaction protein of A-Raf. Hopefully, all of these interaction proteins may contribute to a better understanding of regulation of Raf isoforms and their selective role in cellular regulation.
Our data presented in this work suggest that TH1 binds preferentially with activated forms of A-Raf kinase as shown in Fig. 3. A-Raf can be activated by serum alone or in combination with upstream oncogenic Ras and Src stimulation. From its ability to phosphorylate MEK1 substrate as indicated in Fig. 5, the active forms of A-Raf show increased binding with TH1 (Fig. 3). In this situation, A-Raf enzymatic activity was inhib-  5). Total DNA was kept at 4 g/transfection with pcDNA3.1-myc empty vector. After 48 h, cells were serum starved for 18 h before restimulation by serum. Untreated (lanes  1, 2, and 4) and treated (lanes 3 and 5) cells were collected and lysed as described under "Experimental Procedures" and then precipitated (IP) by anti-myc antibody plus protein G-agarose. The precipitates were analyzed by Western blotting using anti-A-Raf antibody. The expression of A-Raf (middle panel) and myc-TH1 (bottom panel) was confirmed by immunoblotting (IB) one-tenth of the cell lysates used in the binding assay. B, binding assay with HEK293 cells. The experiment was performed as above except that HEK293 cells were used for transfection. Control and experimental samples are indicated. C, binding of TH1 with kinasedead A-Raf. Similar experimental procedures were followed except that HAtagged kinase-dead A-Raf plasmid was used for transfection. D, direct comparison of interaction with TH1 by wild type A-Raf and kinase-dead A-Raf. HA-tagged wild type and kinase-deadA-Raf were detected by immunoblotting as indicated. ited by overexpression of TH1 either upon serum stimulation or upon upstream oncogenic Ras/Src stimulation as shown in Figs. 4 and 5. Taken together, the enhanced binding of TH1 with activated forms of A-Raf contributes to its down-regulation on A-Raf enzymatic activity. Thus, TH1 functions as a negative regulator of A-Raf to avoid excessive A-Raf activation in cells. But whether this binding affects the conformation of A-Raf or just brings active A-Raf away from the ERK pathway is still not clear.
Although TH1 can bind directly with A-Raf, little is known about its function at present. Sequence analysis of TH1 protein revealed that multiple protein kinase phosphorylation sites were found. Such phosphorylation sites include several casein kinase 2␤ phosphorylation sites, a protein kinase C phosphorylation site, and two unique phosphorylation sites: tyrosine protein kinase and cAMP/cGMP-dependent protein kinase phosphorylation (indicated in Fig. 2A). In addition to these protein kinase phosphorylation sites, TH1 also contains several N-myristoylation sites, which might be involved in the recruitment of TH1 to membrane for certain interactions and several N-glycosylation sites for further procession. We mapped out the regions in TH1 which are required for A-Raf binding as Nterminal amino acids 1-372. Interestingly, we have observed in an in vitro substrate phosphorylation assay that TH1 could really be phosphorylated by A-Raf, but which protein kinase phosphorylation sites were involved was not verified (data not shown here). Whether this phenomenon correlates with the TH1 function on A-Raf needs to be investigated further. The regulation of TH1 by A-Raf kinase may play a role in other biological activities, which are not clear at present.
Previous investigations have shown that the Raf protein kinase family members display differences in their abilities to FIG. 5. TH1 overexpression suppressed the A-Raf enzymatic activity stimulated by upstream oncogenic Ras and/or Src. COS-1 or HEK293 cells were transiently transfected with 3 g of myc-tagged TH1 plasmid, 0.5 g of oncogenic Ras plasmid, 0.5 g of oncogenic Src plasmid as indicated. Total DNA was kept at 4 g with empty vector. After 48 h, cell lysates were prepared and quantitated. 500 g of individual cell lysate was used for in vitro A-Raf kinase assay. The endogenous A-Raf and myc-tagged TH1 protein levels were indicated by Western blotting (IB). control cell cycle G 1 depending on their effects on both ERK signal intensity and duration (67). Low levels of Raf activity can promote cell cycle progression, whereas high levels of Raf activity can induce cell cycle arrest. Other studies also indicated that A-Raf played an important role in cell cycle progression and proliferation in vascular smooth muscle cells (24) and in hematopoietic cells (23). Recent studies have shown that in murine fibroblast cells only moderate Raf activity is necessary for G 1 progression and cell proliferation such as C-Raf and A-Raf, whereas potent Raf activation such as B-Raf activation may lead to a p21 Cip1 -mediated cell cycle arrest. The activation of either ⌬B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of ⌬A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to ␤-estradiol (21). Based on these studies, our data showed that TH1 specifically suppressed A-Raf enzymatic activity, and this may contribute to the cell cycle arrest observed.
In summary, we have confirmed that TH1 serves as a new specific negative regulator of A-Raf kinase and is involved in Ras/Raf/MEK1 signal pathway. Its effects on A-Raf kinase provide us a clue to the understanding of Raf isoform-specific regulation in those cells and the unique function of A-Raf on cell cycle progression through ERK signal pathway, but the exact mechanism underlying its effects on A-Raf kinase needs to be elucidated further.