INTRODUCTION

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Recent studies have advanced significantly our understanding of how TGF¹ superfamily members mediate their biological effects. The discovery of TGF receptors and Smad proteins along with recent insights into the mechanism of their activation have allowed us to trace a TGF signal transduction pathway from the cell membrane to the nucleus (reviewed in Refs. 1 and 2). TGF family members initiate signaling at the cell surface by binding and bringing together two different but related serine/threonine kinase receptors, type I and type II. First, the ligand binds to the type II receptor, which recruits and transphosphorylates the type I receptor on the GS domain, a region in the juxtamembrane domain that is rich in serine and glycine residues (3). A mutation in the GS domain can lead to constitutive activation of the receptor (4) and such an activated receptor mimics the effects of the entire receptor-ligand complex in the absence of growth factor and the type II receptor. Thus, the type I receptor is considered the primary transducer of signals to downstream components of the pathway. So far, seven type I receptors or activin receptor-like kinases (ALK1-7) have been identified in vertebrates (reviewed in Refs. 2 and 5).

Once the type I receptor is activated, it associates with specific receptor-regulated Smad proteins and phosphorylates them on the last two serine residues on the carboxyl-terminal domain (6-9). Smad proteins are essential components of TGF signaling that link ligand/receptor signals to transcriptional control (10-16). All members possess two highly conserved MAD homology domains in the amino (MH1) and carboxyl (MH2) terminus that are connected by a proline-rich nonconserved region (reviewed in Refs. 2, 17, and 18). Once receptor-regulated Smad proteins are phosphorylated they can associate with a common partner, Smad4, and translocate to the nucleus. Once there, they may associate with specific DNA-binding proteins in order to generate transcriptional complexes, which subsequently will activate specific target genes. For instance, TGF and activin regulate the Xenopus genes Mix.2 (13), XFKH1 (19), and goosecoid (20), whereas the Xenopus genes Xom (21), Xvent-2 (22), and Msx-1 (23), and the mouse homeobox gene Tlx2 (24) are regulated by BMP signals.

Biochemical and biological studies have established at least two distinct TGF family pathways: one shared by TGF and activin and the other by the BMP2 and 4 subfamily. Characterization of these pathways has shown that a given ligand can induce the formation of heteromeric complexes between different receptors and that a given receptor can recognize different ligands and substrates, suggesting a certain redundancy or cooperativity in signaling. Thus, TRI (ALK5) in concert with TRII can transduce the TGF responses (25, 26), whereas ActRIB (ALK4) acting in union with ActRII or ActRIIB transduce the same signals but in response to activin (26, 27). Consistent with these findings, TRI and ActRIB, which display 90% identity between their kinase domains, can signal through the same downstream components, Smad2 and 3, to generate the same set of responses (6, 28, 29). Thus, TGF and activin illustrate a case of convergent signaling. A similar scenario is displayed by the BMP subfamily. In this case, BMP2/4 bind BMPRII in concert with ALK3 or ALK6 (30, 31) and activate Smad1 and 5 signaling pathways (7, 32, 33). The existence of a plausible third pathway shared by activin and BMP7 has been suggested, since both ligands can bind ActRII and IIB and recruit the type I receptor, ALK2. Hence, ALK2 could represent a candidate receptor that mediates common responses for these factors.

ALK2, also termed ActRI, SKR1, or Tsk-7L, was initially identified as an activin type I receptor because of its ability to bind activin in concert with ActRII or IIB. However, it was not demonstrated that such ligand-receptor complexes were active and could transduce signals under physiological conditions (34-36). Later, it was reported that a specific antisera to ALK2 receptor could immunoprecipitate both a BMP7, as well as an activin-receptor complex. This suggested that ALK2 might indeed mediate common responses for activin and BMP7 (37, 38) and depicted another example of receptor sharing and convergent signaling. However, the recent studies in early Xenopus embryos using a constitutively active form of the ALK2 receptor has shown that ALK2 generates signals similar to BMPs and that it cannot mimic activin signals in the early embryo (23, 39, 40).

In order to characterize ALK2 function we investigated the signaling pathway regulated by this receptor. In the present study, we show that BMP7 and activin stimulate different pathways in the BMP and activin responsive cell line, P19. Although, both factors bind ActRII and IIB, BMP7 specifically binds endogenous ALK2 whereas activin binds ALK4. Stimulation of P19 cells with BMP7 leads to phosphorylation of endogenous Smad1 and Smad5 and association with endogenous Smad4 as well as to induction of the promoter of the homeobox gene, Tlx2. In the same cells, activin signals through endogenous Smad2 and Smad4 leading to induction of a specific activin target gene. Thus, the ALK2 receptor appears to signal through Smad1 and 5 since an activated form of ALK2 mimics BMP7 but not activin signals in P19 cells. Moreover, activated ALK2 also induces the phosphorylation of Smad1, 5, and 8, and the association of Smad1 with Smad4 in transfected COS-1 cells. Therefore, we provide evidence that ALK2 functions as a BMP type I receptor and is likely to transduce BMP7 but not activin signals. In addition, we show that the orphan type I receptor ALK1 (TSRI) which is most closely related to the ALK2 receptor (34) also activates Smad1-dependent signaling.

	EXPERIMENTAL PROCEDURES

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Construction of Expression Vectors-- The construction of pCMV5B/Flag-Smad1, Flag/Smad2, and Flag/Smad2(3SA) has been described previously (6, 32, 41). The mutant Flag/Smad1(458), where the last 7 residues at COOH-terminal tail were deleted, was generated by a polymerase chain reaction-based strategy and was fully sequenced. The construction of His/Smad1(MH2) domain, for bacterial expression, was performed by subcloning the MH2 domain of Smad1 (amino acids 242-465) into the pRSETB expression vector encoding an amino-terminal hexahistidine tag (Invitrogen). The Smad3, Smad5, and Smad8 constructs were provided by R. Derynck (University of California, San Francisco, CA), J. M. Yingling (Duke University, Durham, NC), and W. Vale (The Salk Institute, San Diego, CA), respectively. The ALK2 and ALK1 receptors were described previously (34) and the constructs ALK2/HA (Q207D) and ALK1/HA (Q201D) were generated by a polymerase chain reaction-based strategy and were fully sequenced. The construction of pTLX2-lux and pA3-lux reporter genes has been described previously (24, 42), respectively.

Preparation of Polyclonal Antisera-- Bacterial expression constructs for Smad1, 2, and 4 were constructed by subcloning the non-conserved region from Smad1, 2, or 4 into pGEX-4T1. The glutathione S-transferase fusion proteins generated were used as antigens for rabbit polyclonal antisera production. Specific type I receptor antisera obtained as described previously (43) were kindly provided by Dr. Kohei Miyazono (The Cancer Institute, Tokyo, Japan) and Dr. P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden) and ActRII and IIB specific antisera were a generous gift of Dr. W. Vale (The Salk Institute, San Diego, CA).

Cell Lines and Transfections-- P19 cells were cultured in -minimal essential medium containing 7.5% calf serum and 2.5% fetal bovine serum, and were transiently transfected using the calcium phosphate-DNA precipitation method. MC3T3-E1 derived by Sudo et al. (44) were maintained in -minimal essential medium + 10% fetal bovine serum and were obtained from Dr. J. Aubin (University of Toronto, ON). COS-1 cells were grown in Dulbecco's modified Eagle's medium containing high glucose and 10% fetal bovine serum. COS-1 cells were transiently transfected with the indicated vectors using a DEAE-dextran method as described previously (32).

Immunoprecipitations and Immunoblotting-- For endogenous Smads, endogenous type I and type II receptors and anti-HA immunoprecipitations, cell were lysed in lysis buffer (45) subjected to immunoprecipitation with specific Smad antisera, specific type I and II receptors antisera or anti-HA antibody, respectively, followed by adsorption to protein A-Sepharose (Pharmacia). For endogenous Smad immunoprecipitations, cell lysates were precleared with protein A-Sepharose beads and preimmune sera for 30 min. For -Flag or -Myc immunoprecipitations cell lysates were subjected to immunoprecipitation with anti-Flag M2 antibody (IBI, Eastman Kodak) or with 9E10 mouse anti-Myc antibody, respectively, followed by adsorption to protein G-Sepharose (Pharmacia) as described previously (32). Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For determination of Flag, HA, or Myc-tagged Smad protein levels and endogenous Smad protein levels, aliquots of total cell lysates were separated by SDS-PAGE and assayed by immunoblotting as described previously (32).

Transcriptional Response Assay-- For BMP or activin-inducible luciferase assays, P19 cells were transiently transfected with the reporter plasmid pTlx2-lux or pA3-lux, respectively, and pCMV-gal. Cells were seeded at 20% confluency in 24-well plates and transfected overnight with 0.5-1 µg of DNA per well using the calcium phosphate-DNA precipitation method as described previously (32). To induce the luciferase reporter, cells were treated overnight with the appropriate ligands, lysed, and luciferase activity was measured using the luciferase assay system (Promega) in a Berthold Lumat LB 9501 luminometer. -Galactosidase activity was measured as described previously (8) and data were used to determine transfection efficiency.

Phosphate and Metabolic Labeling-- For [³²P]phosphate labeling, cells were washed and preincubated with phosphate-free medium containing 0.2% dialyzed fetal bovine serum. The cells were then incubated with media containing 1 mCi/ml [³²P]PO₄ for 2 h at 37 °C as described previously (45). For ³⁵S-metabolic labeling, cells were washed and then preincubated with methionine-free medium containing 50 µCi/ml [³⁵S]methionine for 2 h at 37 °C as described previously (45). Cells were then lysed and subjected to immunoprecipitation with specific antibodies. Labeled proteins were analyzed by SDS-PAGE and autoradiography.

Affinity Labeling-- P19 cells or transiently transfected COS-1 cells were affinity labeled using [¹²⁵I]BMP7, [¹²⁵I]BMP2, or [¹²⁵I]activin in media containing 0.2% fetal calf serum at 37 °C for 30 min and the receptors were cross-linked to the ligand as described previously (46). Cells were lysed in lysis buffer containing 10% glycerol as described previously (45). In order to study specific ligand/receptor binding, lysates were immunoprecipitated using specific type I and II receptors polyclonal antisera. For determining Smad-receptor complex interactions, lysates were immunoprecipitated using anti-Flag M2 monoclonal antibody as described previously (6). Complexes were analyzed by SDS-PAGE and autoradiography.

In Vitro Kinase Assay-- Receptor complexes were immunoprecipitated from transiently transfected COS-1 cell lysates as described above except that immunoprecipitates were washed three times with TNTE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, and 1 mM EDTA) followed by two washes in kinase buffer. Receptor complexes were incubated for 30 min at room temperature in kinase buffer containing His/MH2 domain of Smad1 protein which was expressed in bacteria. Briefly, the His/MH2 domain of Smad1 was expressed in BL21(DE3) cells and purified from isopropyl-1-thio--D-galactopyranoside-induced bacterial cultures. The protein was collected on Ni-NTA agarose beads, washed with TNT buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 0.5% Triton) containing 40 mM imidazole, and eluted with 250 mM imidazole. The eluted fusion protein was dialyzed overnight, concentrated, and 2 µg used as a substrate in the kinase assay as described previously (6). Protein phosphorylation was analyzed by SDS-PAGE and autoradiography.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

Characterization of BMP and Activin Receptors on P19 Cells-- To investigate BMP7 signaling we used P19 cells which respond to both BMPs and activin. To characterize the profile of BMP7 receptors, P19 cells were affinity labeled using [¹²⁵I]BMP7 and receptor complexes were immunoprecipitated using specific antibodies directed against various type I and type II receptors. BMP7 receptor complexes were detected on P19 cells and these were found to be composed of the previously characterized type II receptors, ActRII and ActRIIB, together with the type I receptor, ALK2. No cross-linking of BMP7 to ALK4 was detected, consistent with previous conclusions that ALK4 is an activin-specific type I receptor (26, 27). Since previous studies indicated that ALK2 may bind activin under some conditions (34), we also explored the profile of activin receptors expressed on these cells. Affinity labeling with ¹²⁵I-activin, followed by immunoprecipitation with antibodies to ALK4 or ALK2 revealed the presence of complexes of either ActRII or ActRIIB together with ALK4. However, no interaction of activin with ALK2 was detected, suggesting that in P19 cells ALK2 functions predominantly as a BMP7 receptor (Fig. 1A).

View larger version (63K): [in this window] [in a new window]   Fig. 1.   BMP7 specifically binds the endogenous type I receptor ALK2 in P19 cells. P19 cells were affinity labeled with [125I]BMP7, [125I]activin (A) or with [125I]BMP2 (B). Endogenous receptor complexes were immunoprecipitated (IP) with specific antisera ActRII (II), ActRIIB (IIB), ALK2 (A2), ALK3 (A3), ALK4 (A4), or ALK6 (A6). Ligand-receptor complexes were analyzed by SDS-PAGE and autoradiography from either total (T) or immunoprecipitated cell lysates.

BMP7 Induces a BMP2-like Response-- To investigate BMP7 responses in P19 cells, we examined regulation of Tlx2, a homeobox gene of the HOX11 class. Previously, we showed that Tlx2 functions during gastrulation in the mouse and is strongly induced by BMP2 (24). Analysis of the Tlx2 gene showed that it contained a BMP-responsive promoter that was induced by BMP2 in P19 cells (24). To characterize BMP7 signaling, pTlx2-Lux was transiently transfected into P19 cells and cultures were incubated in the presence or absence of 10 nM BMP7, 10 nM BMP2, or 2 nM activin prior to analysis of reporter gene activation. Under these conditions, BMP7 induced the Tlx2 promoter 7-fold, similar to the response observed for BMP2 (Fig. 2, TLX2-Lux). In contrast, activin had no detectable effect on the Tlx2 promoter, similar to previous observations (24).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   BMP7 specifically induces the transcription of the Tlx2 reporter gene in P19 cells. P19 cells were transiently transfected with a specific BMP-responsive (pTlx2-lux) or an activin-responsive (pA3-lux) reporter gene. Cells were incubated overnight in the absence (basal) or presence of 10 nM BMP2, 10 nM BMP7, or 2 nM activin. The relative luciferase activity was measured in cell lysates and normalized to -galactosidase activity. Data are expressed as the mean ± S.D. of triplicates from a representative experiment.

BMP7 Leads to Specific Phosphorylation of Smad1 and Smad5-- MAD-related proteins are critical intracellular mediators of TGF superfamily signaling (reviewed in Refs. 2, 17, 18, 50, and 51). To investigate the intracellular pathways activated by BMP7 in P19 cells, we examined the regulation of endogenous Smads in these cells. For these studies, we generated Smad antisera that are specific for different subclasses of Smads. To characterize these antisera, tagged Smad proteins were expressed in COS-1 cells and total cell lysates were immunoblotted using specific Smad antisera. In Fig. 3, we show that anti-Smad1 recognizes Smad1 and the closely related Smad5, anti-Smad2 recognizes Smad2 and Smad3, and anti-Smad4 is specific for Smad4 protein. Although these antisera displayed some cross-reactivity between closely related Smads, both Smad1 and Smad5, and Smad2 and Smad3 exhibited distinct mobilities when analyzed by SDS-PAGE. This allowed us to distinguish these Smads during our subsequent analysis.

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Specificity Smad antisera. COS-1 cells were transiently transfected with vector alone (), Flag/Smad1 (S1), Flag/Smad2 (S2), Flag/Smad3 (S3), Smad4/HA (S4), Smad7/HA (S7), or Myc/Smad8 (S8) (A) or with vector alone (), Flag/Smad1 (S1), Flag/Smad2 (S2), or Flag/Smad5 (S5) (B). Total cell lysates were immunoblotted with specific antibodies for Smad1 (Smad1 blot), Smad2 (Smad2 blot), or Smad4 (Smad4 blot). Expression of Smad proteins was determined by immunoblotting total cell lysates using anti-Flag, anti-Myc, and anti-HA monoclonal antibodies as indicated.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of endogenous Smad proteins after stimulation by BMP7, BMP2, and activin in P19 cells. P19 cells were labeled with [32P]phosphate or [35S]methionine and then incubated for 30 min in the absence (control) or presence of 10 nM BMP2, BMP7, or activin. Endogenous Smad proteins were immunoprecipitated (IP) with preimmune (P), Smad1 (S1), Smad2 (S2), or Smad4 (S4) antisera and analyzed by SDS-PAGE and autoradiography (A, B, and D). The migration of Smad1 or 5 in Smad1 IPs is indicated to the left, while Smad2 and Smad3 in Smad2 IPs is shown on the right. For examination of time-dependent phosphorylation of Smad1 and Smad5 cells were treated with BMP2 or BMP7 for the indicated times (C).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   BMP7 induces Smad1 and Smad4 association in P19 cells. P19 cells were incubated with 10 nM BMP2 (B2), BMP7 (B7), or activin for 1 h. Cell lysates were immunoprecipitated (IP) with preimmune (P), Smad1 (S1), Smad2 (S2), or Smad4 (S4) antisera and then immunoblotted with Smad4 antisera (Smad4 blot). The migration of Smad4 is indicated. To determine the level of endogenous Smad4 expression, total cell lysate was immunoblotted with Smad4 antisera.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   ALK2 receptor induces phosphorylation of Smad1, 5, and 8 and transcription of the Tlx2 gene. A, phosphate-labeled MC3T3-E1 cells were incubated in the absence (control) or presence of 10 nM BMP7 or activin for 30 min. Endogenous Smad proteins were immunoprecipitated (IP) from cell lysates with preimmune (P), Smad1 (S1), or Smad2 (S2) antisera. Labeled proteins were analyzed by SDS-PAGE and autoradiography (32PO4). B, ALK2 induces the phosphorylation of Smad1, 5, and 8. COS-1 cells were transiently transfected with empty vector alone () or combinations of wild type (WT) or activated ALK2 receptor (A*) together with wild type or mutant Flag/Smad1 (458), or with wild type Flag/Smad2, untagged Smad5, or Myc/Smad8. Cells were labeled with [32P]phosphate and Flag/Smad1 or Flag/Smad2 were purified by immunoprecipitation with anti-Flag M2 antibody, whereas untagged Smad5 was purified with anti-Smad1 polyclonal antibody and Myc/Smad8 with monoclonal anti-Myc antibody and analyzed by SDS-PAGE and autoradiography (32PO4). C, ALK2 receptor specifically stimulates Tlx2 transcription. P19 cells were transiently transfected with vector alone (pCMV5) or with wild type (WT) or activated ALK2 (A*), ALK6 (A*), or ALK4 (A*) receptors. The relative luciferase activity was measured in cell lysates and normalized to -galactosidase activity. Data are expressed as the mean ± S.D. of triplicates from a representative experiment.

ALK2 Activates Smad1 and Induces BMP-specific Signals-- To directly assess the regulation and activation of Smads by ALK2 we substituted the glutamine residue at position 207 with an aspartate residue. Previous studies have shown that this type of mutation constitutively activates the kinase domain of type I Ser/Thr kinase receptors (4). Expression of constitutively active ALK2 together with wild type Smad1 resulted in a strong induction of Smad1 phosphorylation (Fig. 6B), and association of Smad1 with Smad4 (data not shown). This regulation was specific, since Smad2 phosphorylation was unaffected by the constitutively active ALK2. Since phosphorylation of receptor-regulated Smads occurs on the COOH-terminal SSXS motif (6-9), we sought to determine whether ALK2 might similarly regulate Smad1. In contrast to wild type Smad1, activated ALK2 was unable to induce phosphorylation of Smad1(458). Since this mutation removes the COOH-terminal serines, these results suggest that the SSXS motif of Smad1 is the site for ALK2-dependent phosphorylation of the protein. We also tested whether activated ALK2 could induce phosphorylation of Smad5 and Smad8. Consistent with our observations in P19 cells and previous reports, both Smad5 and Smad8 were efficiently phosphorylated by the activated receptor (Fig. 6B, lower panels).

Smad1 Is a Substrate for ALK2-- To determine whether Smad1 directly interacts with BMP7 receptor complexes, we expressed Smad1 or Smad1(458) together with ActRIIB and either wild type or kinase-deficient ALK2. BMP7 receptor complexes were then affinity labeled with [¹²⁵I]BMP7 and complexes coprecipitating with Smad1 analyzed by SDS-PAGE and autoradiography. Similar to our previous observations on the TGF receptor (6), no association between wild type Smad1 and wild type BMP7 receptors could be detected. However, analysis of complexes composed of kinase-deficient type I receptor showed coprecipitation of BMP7-labeled receptors with Smad1. Furthermore, introduction of a phosphorylation site mutation into Smad1 allowed efficient coprecipitation of wild type BMP7 receptors with Smad1. These data are consistent with the notion that Smad1 transiently associates with activated ALK2 and that phosphorylation leads to dissociation of the Smad1 substrate from the receptor. In none of these experiments were we able to detect associations between BMP7 receptors and either wild type Smad2 or the phosphorylation site mutant, Smad2(3SA) (Fig. 7A).

View larger version (60K): [in this window] [in a new window]   Fig. 7.   ALK2 receptor specifically interacts with and phosphorylates Smad1 protein. A, ALK2 interacts with Smad1. COS-1 cells were transiently transfected with either wild type (WT) or mutant Flag/Smad1 (458) or Flag/Smad2 (3SA) and the indicated combination of wild type or kinase-deficient (KR) ALK2 together with ActRIIB receptors. Cells were affinity labeled with 2 nM [125I]BMP7 and cell lysates were subjected to immunoprecipitation with anti-Flag M2 antibody. Receptor complexes were visualized by SDS-PAGE and autoradiography (Flag IP). To confirm receptor expression, aliquots of total cell lysates were analyzed by SDS-PAGE (receptors). Expression of Flag/Smad proteins was determined by immunoblotting total cell lysates using anti-Flag M2 antibody (Flag blot). B, in vitro phosphorylation of Smad1 by ALK2. COS-1 cells were transiently transfected with the indicated combination of type II, HA-tagged (ActRII or IIB) or Flag-tagged (BMPRII), and untagged type I receptors (ALK2 or ALK6), or with wild type or activated (A*) ALK2/HA alone. Cells were incubated with 10 nM BMP2 or BMP7 for 30 min at 37 °C, lysed, and receptor complexes isolated by immunoprecipitation using anti-HA antibody. Receptor complexes were incubated in a kinase assay buffer containing [-32P]ATP and bacterially expressed His/MH2 domain of Smad1 as a substrate. Smad1(MH2) phosphorylation was visualized by SDS-PAGE and autoradiography (in vitro kinase). The Coomassie-stained gel indicating constant levels of Smad1(MH2) is shown.

The Orphan ALK1 Receptor Also Activates Smad1-dependent Signaling-- Comparison of the kinase domains of type I receptors indicates that ALK2 is more closely related to ALK1 (80% identity) than it is to ALK3 and ALK6. ALK1 (TSRI) was initially identified as a TGF receptor due its ability to bind TGF when overexpressed with TRII (34). However, no TGF mediated signals have been characterized and its endogenous ligand has not been identified. Since ALK1 is most closely related to ALK2, and given that ALK2 activates Smad1 signaling, we examined whether Smad1 is also a target for ALK1. To investigate this, we constructed a constitutively active version of ALK1 by replacing the glutamine at position 201 with an aspartate. The capacity of wild type and activated forms of ALK1 to activate Smad1 was then assessed in transiently transfected COS-1 cells. Expression of constitutively active ALK1 together with wild type Smad1 or Smad2 resulted in a strong induction of Smad1 phosphorylation, but not Smad2, similar to that observed for ALK2 (Fig. 8A), whereas TRI specifically induced Smad2 phosphorylation (Fig. 8A). We also found that like ALK2, ALK1 also induced phosphorylation of Smad5 (data not shown). To examine whether ALK1 might also activate Smad1-dependent signaling pathways, we examined the regulation of pTlx2-Lux and pA3-Lux by the constitutively active ALK1 in P19 cells. Expression of activated ALK1 in P19 cells induced the Tlx2 promoter to a level similar to that observed for ALK2 (Fig. 8B), whereas activated ALK1 was unable to induce the activin-responsive reporter gene, pA3-lux (data not shown). Thus, the orphan receptor ALK1 like ALK2, also appears to function to regulate BMP signaling.

View larger version (47K): [in this window] [in a new window]   Fig. 8.   ALK1 type I receptor activates Smad1-dependent signaling. A, ALK1 induces Smad1 phosphorylation. COS-1 cells were transiently transfected with wild type Flag/Smad1 or Flag/Smad2 in combination with wild type (WT) or activated ALK1 (A*), ALK2 (A*), or TRI (A*) receptors. Cells were labeled with [32P]phosphate and Flag/Smad1and Flag/Smad2 were purified by immunoprecipitation with anti-Flag M2 antibody and analyzed by SDS-PAGE and autoradiography (32PO4). Expression of Flag/Smad1 and Flag/Smad2 protein was determined by immunoblotting total cell lysates using anti-Flag-M2 antibody (Flag blot). B, ALK1 induces transcription of BMP-responsive gene Tlx2. P19 were transiently transfected with vector alone (pCMV5) or with wild type (WT) or activated ALK1 (QD) or ALK2 (QD) receptors. Data is plotted as described in the legend to Fig. 2.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The characterization of BMP and activin signaling has shown that these factors induce formation of specific heteromeric combinations of type I and type II Ser/Thr kinase receptors. For example, BMPs bind the type II receptor BMPRII in concert with the type I receptors, ALK3 or ALK6, and activin promotes the association of the type II receptors, ActRII or IIB, with the type I receptor, ALK4 (ActRIB). In contrast to these specific type I receptors, ALK2 has been observed to bind activin in concert with ActRII and ActRIIB when overexpressed in COS cells (34), as well as mediating binding of BMP7 in cell lines that express endogenous ALK2 (37). Therefore, it has been proposed that ALK2 is a shared receptor for activin and BMP7 and that it might be responsible for mediating common responses to these factors (38). Thus, the goal of the present study was to characterize the signaling pathways activated by ALK2 and to investigate the potential function of this receptor in mediating responses to BMP7 and activin. Using P19 cells, we show that BMP7 and activin bind to the common type II receptors, ActRII and IIB, but recruit different type I receptors into the ligand-receptor complex and activate distinct Smad signaling pathways. Thus, BMP7 signaling through the ALK2 receptor can activate Smad1 and 5 and induce the BMP-responsive homeobox gene, Tlx2, whereas activin, through ALK4 activates Smad2 to induce the ARE promoter. Furthermore, we show that ALK2 associates with Smad1, mediates phosphorylation of Smad1 on its COOH-terminal SSXS motif and that activated forms of the receptor can mimic BMP-dependent signaling in P19 cells. ALK2 also induces phosphorylation of Smad5 and Smad8 and we show that the orphan ALK1 receptor, which is most closely related to ALK2, also activates Smad1. Thus, ALK2 can mediate BMP7-dependent signaling and does not appear to encode a functional receptor for activin.

BMP and Activin Receptors-- P19 cells are an embryonic carcinoma cell line that are ideal for investigating the function of activin and BMP signaling pathways. These cells are BMP and activin-responsive and express endogenous BMP type I receptors, such as ALK3 and ALK6 (53) as well as ALK2 (36). These cells also express activin receptors that have not previously been characterized (52). In the present study, we show that BMP2, in addition to BMP7 and activin, bind endogenous ActRII and IIB in P19 cells. In addition to these type II receptors, P19 cells also express BMPRII (53) that interacts with BMP2 and 7 but not activin (31, 54). Thus it is likely that several type II receptors can function in BMP signaling (Fig. 9A). It is interesting to note that previous studies in Drosophila which demonstrated that the type II receptor PUNT, which is highly related to ActRII and binds activin with high affinity, only binds BMP2 when the appropriate type I receptor is coexpressed (49). In COS-1 cells, we have observed a similar behavior for BMP2 binding to the mammalian activin type II receptors,² and previous studies on GDF5 have shown that this ligand binds ActRII or IIB in union with ALK6 (55). All of this suggests that ActRII and IIB have broad specificity and function as type II Ser/Thr kinase receptors for numerous TGF family members. This is supported by functional studies in Xenopus that show that a truncated ActRII or IIB receptor blocks both activin and BMP signaling in vivo (38, 56-58) whereas such cross-reactivity does not occur for a truncated BMPRII, which only binds BMPs (59). It is interesting that the broad specificity of activin type II receptors is conserved in Drosophila, raising the possibility that overlapping specificity may be of functional relevance during development, perhaps by allowing different ligands to regulate each other by competing for common receptor subunits.

View larger version (30K): [in this window] [in a new window]   Fig. 9.   The type I receptor determines the nature of the biological response. A, BMP2/4, BMP7, and activin, representing different subgroups of the TGF superfamily, can share the type II receptors ActRII and IIB. In contrast, BMPRII only binds BMPs. However, the specificity of the signal is determined by the type I receptor recruited by the ligand-type II receptor complex. Thus, BMP2/4 signal primarily through ALK3 or ALK6, BMP7 signals through ALK2, and ALK4 transduces activin signals. Smad1, Smad5, and likely Smad8 function downstream of BMP type I receptors and participate in the induction of the homeobox gene, Tlx2. Smad2 transduces activin receptor (ALK4) signals and mediates activation of the activin response genes, ARE (Mix.2) and TARE (gscd), while Smad3 (starred) is similarly activated but blocks FAST2-dependent transcription (see Ref. 60). B, comparison of the L45 loop within the kinase domain of the type I receptors indicates that this family of receptors can be divided into three groups. The first group is comprised of ALK3, ALK6, and the Drosophila, TKV receptor. The second group, in which the L45 loop is completely conserved, is comprised of ALK4, ALK5, ALK7, and Drosophila, ATR-I. The third group consists of ALK1, ALK2, and Drosophila, SAX receptor. The position of the L45 loop sequence for ALK3, TRI, and ALK2 receptors is indicated.

ALK2 Mediates BMP7 but Not Activin Signaling-- Considerable work in Xenopus has defined distinct biological responses to BMPs and activin/TGF. For example, both factors are implicated in the induction of mesoderm, but BMPs act as mesoderm ventralizers, whereas activin induces dorsal mesoderm (62, 63). In addition, other studies have shown that the expression of dominant negative versions of BMP7 and BMP2/4 induce neuroectoderm in animal caps, whereas dominant negative versions of activin do not mimic this phenotype (64). Similarly, both BMP2 and 7 have inhibitory effects on neurogenesis in Xenopus (65). In early mammalian development this distinction between BMP and activin signaling pathways has not been defined. P19 cells have been used as a model for studying mesodermal and neural differentiation in the mouse. BMP2/4 and activin induce mesoderm markers (66) and inhibit retinoic acid-induced neural differentiation in these cells (67, 68). However, while BMPs induce apoptosis in combination with retinoic acid (67, 69), activin functions as a mitogen of undifferentiated P19 cells (70). However, the mechanisms underlying these complex biological responses are poorly defined.

Convergent Signaling to Smads 1, 5, and 8 by BMPs-- The specific regulation of Smad1 signaling by ALK2 is analogous to the previously described role for Smad1 as a downstream target of BMP2 signaling through ALK3 and ALK6 (1, 7). Indeed, in our current studies, the time courses and magnitudes of Smad1 activation by BMP2 or 7 were indistinguishable. These results suggest that there is considerable convergence in BMP signaling. Thus, multiple BMPs functioning through a limited subset of cell surface receptors may all activate a common downstream signaling pathway to elicit shared biological responses (Fig. 9A). Apart from Smad1, two other receptor-regulated Smads, Smad5 and 8, are likely to participate in this pathway. Smad5 was previously characterized in the BMP pathway (71), while Smad8 was recently isolated and shown to be regulated by constitutively activated ALK2 (39). Thus, ALK1, ALK2, ALK3, and ALK6 all likely activate this common set of Smads and appear to possess considerable overlap in function. Interestingly, BMP7 can also bind ALK6 in some cells and in one cell line has been observed to bind ALK3. This raises the possibility that in addition to utilizing ALK2, BMP7 can activate Smad1, 5, or 8 through other type I receptors. This convergence could suggest a remarkable degree of overlap in ligand function during development. While a certain amount of redundancy is likely to occur, the restricted expression patterns of ligands during development may ensure that many of them fulfill precise roles at specific places and times to control patterning and tissue remodeling. Moreover, cell type specific responses to BMPs may be controlled by the regulated expression of distinct Smad1 nuclear targets. Thus while Smad1, 5, and 8 activation may be a common pathway for a large number of BMP ligands, the complex nature of the biological response to BMPs may be dictated by an array of nuclear targets.

Receptor Determinants of Smad Targetting-- Since the identity of the type I receptor in the ligand-receptor complex determines the nature of the signal, the study of the structural requirements in the kinase domain that determine downstream signaling specificity has received considerable attention. Recently, a structural analysis of type I receptor kinase domains has led to the identification of a 9-amino acid region, termed the L45 loop, which seems to be critical in specifying the signal. Thus, replacing the L45 loop in ALK2 with that from TRI, conferred on the chimeric type I receptor the capacity to signal TGF-like responses (74). This loop is predicted to be located on an exposed region of the kinase domain between subdomains IV and V and thus could represent a Smad-docking site.