Recombinant expression and purification of smad proteins.

Smads transduce intracellular signals initiated by members of the transforming growth factor beta (TGF beta) family, including activins, TGF betas, and bone morphogenetic proteins. Recently, various models concerning the mechanism of Smad action have been proposed; however, these models are basically qualitative. Quantitative verification of the validity of the models requires significant amounts of purified Smad proteins, but purification of full-length Smad protein has not been straightforward even using recombinant protein expression systems. Here, we report purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydroxylapatite columns, expressed Smads were purified to near homogeneity as judged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 100 microg/l culture for Smad4. The purified Smad proteins had three known in vitro activities: Smad2 phosphorylation by TGF beta receptor complexes immunoprecipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and Smad2 interaction with calmodulin. The data suggest that purified proteins could be useful for biochemical analyses to evaluate the current models quantitatively.

Identification and characterization of constitutively active Smad2 mutants: evaluation of formation of Smad complex and subcellular distribution.

Smads mediate activin, transforming growth factor beta (TGFbeta), and bone morphogenetic protein signaling from receptors to nuclei. According to the current model, activated activin/TGFbeta receptors phosphorylate the carboxyl-terminal serines of Smad2 and Smad3 (SSMS-COOH); phosphorylated Smad2/3 oligomerizes with Smad4, translocates to the nucleus, and modulates transcription of defined genes. To test key features of this model in detail, we explored the construction of constitutively active Smad2 mutants. To mimic phosphorylated Smad2, we made two Smad2 mutants with acidic amino acid substitutions of carboxyl-terminal serines: Smad2-2E (Ser465, 467Glu) and Smad2-3E (Ser464, 465, 467Glu). The mutants enhanced basal transcriptional activity in a mink lung epithelial cell line, L17. In a Smad4-deficient cell line, SW480.7, Smad2-2E did not affect basal signaling; however, cotransfection with full-length Smad4, but not transfection of Smad4 alone, resulted in enhanced basal transcriptional activity, suggesting that the constitutively active Smad2 mutant also requires Smad4 for function. In vitro protein interaction analysis revealed that Smad2-2E bound more tightly to Smad4 than did wild-type Smad2; dissociation constants were 270 +/- 66 nM for wild-type Smad2:Smad4 complexes and 79 +/- 18 nM for Smad2-2E:Smad4 complexes. Determination of the subcellular localization of Smad2 revealed that a greater percentage of Smad2-2E was localized in the nucleus than wild-type Smad2. These results suggest that Smad2 phosphorylation results in both tighter binding to Smad4 and increased nuclear concentration; those changes may be responsible for transcriptional activation by Smad2.

Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages.

The role of activin, a dimer of inhibin beta subunit, in mouse peritoneal macrophages was evaluated. Activin activity in the cultured macrophages was augmented in response to activation by LPS. In Western blot analysis, immunoreactive activin A was detected in the culture medium only when the macrophages were stimulated by LPS. Although mRNA expression of betaA subunit was detected, that of alpha and betaB subunit was not found in macrophages by reverse RT-PCR. The activin betaA mRNA level was increased in macrophages by LPS, suggesting that the activin production augmented by LPS is regulated at the mRNA level of the betaA gene. The mRNAs of four activin receptors (ActRI, ActRIB, ActRII, and ActRIIB) were also detected in the peritoneal macrophages, and the mRNA levels, except for ActRIB, were decreased during the LPS treatment. Exogenous activin A stimulated the mRNA expression and gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) in macrophages in both the presence and the absence of LPS. In contrast, activin did not affect the production of MMP-9 in macrophages. These results suggested that 1) mouse peritoneal macrophages produced activin A; 2) expression of activin A was enhanced with activation of the macrophages; 3) the macrophages also expressed activin receptors; and 4) exogenous activin A stimulated MMP-2 expression and activity, implicating activin A as an positive regulator of MMP-2 expression. Considering that MMP-2 constitutes the rate-limiting proteinase governing the degradation of basement membrane collagens, activin A may be involved in migration and infiltration of macrophages through the basement membrane in an inflammatory state.

Smad proteins physically interact with calmodulin.

The Smad family of intracellular proteins mediates signals generated by activin and other transforming growth factor beta-related proteins via specific heteromeric complexes of transmembrane receptor serine kinases (1, 2). xSmad2 has been implicated as an activin signal mediator that may participate in transcriptional regulation (3, 4). We have employed an interaction cloning strategy to identify xSmad2-binding proteins and found that calmodulin directly associated with Smads. xSmad2, generated either by in vitro translation or by overexpression in COS cells, specifically bound to calmodulin-agarose; the association was calcium-dependent and required xSmad2 N-terminal residues. In the same assay, xSmad1 and hSmads 2, 3, and 4 also bound to calmodulin-agarose. Furthermore, a calmodulin antagonist, W13, increased expression of the activin-inducible transcriptional reporter, 3TP-Lux, whereas overexpression of calmodulin suppressed this reporter. These observations demonstrate that Smad proteins interact with calmodulin in a calcium-dependent way through conserved N-terminal amino acids and suggest a role for calmodulin in regulating Smad function.

Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling.

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.

Suspiciousness, mental simulation, and norm theory.

Seven studies were conducted to replicate the work of Miller, Turnbull, and McFarland (1989), who tested predictions from norm theory (Kahneman & Miller, 1986). The first three studies with stimulus materials identical to those used by Miller et al. failed to confirm that the ease with which the event might be mentally simulated affected the degree of suspiciousness. In Studies 4, 5, and 6, the improbable events were made objectively more probable, but this did not produce significant results. In the 7th study, the objective probability and attitude toward the target were varied. Although there was a main effect for ease of mental simulation, this effect was produced by only 1 of the 3 vignettes.

A Xenopus type I activin receptor mediates mesodermal but not neural specification during embryogenesis.

Activins and other ligands in the TGFbeta superfamily signal through a heteromeric complex of receptors. Disruption of signaling by a truncated type II activin receptor, XActRIIB (previously called XAR1), blocks mesoderm induction and promotes neuralization in Xenopus embryos. We report the cloning and characterization of a type I activin receptor, XALK4. Like truncated XActRIIB, a truncated mutant (tXALK4) blocks mesoderm formation both in vitro and in vivo; moreover, an active form of the receptor induces mesoderm in a ligand-independent manner. Unlike truncated XActRIIB, however, tXALK4 does not induce neural tissue. This difference is explained by the finding that tXALK4 does not block BMP4-mediated epidermal specification, while truncated XActRIIB inhibits all BMP4 responses in embryonic explants. Thus, the type I and type II activin receptors are involved in overlapping but distinct sets of embryonic signaling events.

Regulation of activin type I receptor function by phosphorylation of residues outside the GS domain.

Activin signals through a heteromeric complex of receptor serine kinases by inducing type II receptor-mediated phosphorylation, and consequent activation, of the type I receptor. Type I receptor phosphorylation occurs at a glycine- and serine-rich site in the juxtamembrane domain; phosphorylation at that site correlates with signaling. Investigation of type I activin receptor mutants impaired for GS domain phosphorylation revealed that, in the presence of elevated amounts of type II activin receptor, GS domain phosphorylation is not required for signaling. The type I receptor showed activin-dependent phosphorylation of several tryptic phosphopeptides, suggesting that phosphorylation of receptor I at sites both within and outside the GS domain is required for full signaling.

Formation and activation by phosphorylation of activin receptor complexes.

Activin is a protein growth and differentiation factor that initiates intracellular events through the activation of a complex of transmembrane protein serine kinases. Two subfamilies of receptor serine kinases, type I and type II, have been identified, and both receptor types may be required to generate a transmembrane signal. Investigation of the interaction between various activin receptors (ActRs) revealed that ActRs I and II could exist in a stable complex and that formation of that complex between transiently overexpressed molecules was not regulated by ligand. Analysis of phosphorylation suggested that activin induced phosphorylation of receptor I, probably at residues within a conserved glycine and serine-rich sequence in the juxtamembrane region referred to as the GS domain. Phosphorylation of the GS domain was dependent upon a functional ActRII. Introduction of an activin type I receptor, ALK4, into the mink lung epithelial cell line, L17, conferred activin responsiveness on those cells. Mutation of specific combinations of serines and threonines in the core sequence of the ALK4 GS domain to alanine rendered that receptor incompetent for signaling. Mutation of the same sets of residues to glutamic acid produced molecules that supported activin signaling but that did not display elevated basal signaling anticipated for a constitutively active receptor. However, mutation of a threonine residue in the carboxy-terminal half of the GS domain, T206, to glutamic acid yielded receptors with constitutive activity. Taken together, these results support a role for phosphorylation of type I ActRs in the generation of a biological signal.

Activin receptors: cellular signalling by receptor serine kinases.

Activins are dimeric growth and differentiation factors which signal through a heteromeric complex of receptor serine kinases. Affinity labelling of activin-responsive cells with 125I-labelled activin A reveals activin-binding species of approximately 50 and 75 kDa, known as the type I and type II receptors, respectively. Molecular cloning has yielded genes encoding two type II receptors (ActRII and ActRIIB) and at least two type I receptors (ALK2 and ALK4). These receptors are members of a larger family of receptor serine kinases, which includes receptors for transforming growth factor beta (TGF beta), bone morphogenetic proteins and the Drosophila protein, decapentaplegic. Receptors I and II form a stable complex after ligand binding, resulting in phosphorylation of receptor I by receptor II. We have begun to identify intracellular targets of these molecules by using the intracellular domains of both ActRIs and ActRIIs as probes in the two-hybrid system, a cloning strategy designed to detect interacting proteins in yeast.

Activin and its receptors during gastrulation and the later phases of mesoderm development in the chick embryo.

We have cloned chick homologues of the type-II activin receptor, which we have designated cActR-IIA and -IIB. Binding assays show that the two receptors are indistinguishable in their ability to bind activin-A, with comparable kds. Injection of mRNAs encoding these receptors into Xenopus embryos causes axial duplications. Expression of both receptors can first be detected in the primitive streak by in situ hybridization. This suggests that these genes may be activated in response to mesoderm induction. In agreement with this, we find that treatment of preprimitive streak chick embryos with activin-A leads to rapid induction of the expression of cActR-IIB. At later stages, cActR-IIA transcripts become localized mainly in the notochord and myotome and cActR-IIB in the dorsal neural tube, proximal-anterior part of the limb bud, sensory placodes, and specific regions of the fore- and midbrain. To test the response of early chick embryonic tissues to activin, we designed a new in vitro assay for differentiation. We find that explants of area opaca epiblast or posterior primitive streak from various stages can respond to activin treatment by differentiating into a variety of mesodermal cell types in a dose-dependent manner. These results suggest that the importance of activin-related signaling pathways is not confined to pregastrulation stages and that these receptors may be involved in mediating the effects of inducing signals during later stages of development of the mesoderm, limbs, and nervous system.

Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis.

We have studied the distribution of activin receptor gene expression in the brain, pituitary, ovary, and testis of the adult rat by in situ hybridization, using probes complementary to the mRNAs encoding the mouse activin receptor subtypes II and IIB (ActRII and ActRIIB). Throughout the brain, ActRII mRNA expression was stronger than that of ActRIIB, and the patterns of expression were similar, although not identical. The most intense sites of activin receptor gene expression were the hippocampal formation, especially the dentate gyrus (ActRII), taenia tecta, and induseum griseum; the amygdala, particularly the amygdaloid-hippocampal transition zone; and throughout the cortical mantle, including the primary olfactory cortex (piriform cortex and olfactory tubercle); other regions of the cortex showing lesser degrees of hybridization included the cingulate cortex, claustrum, entorhinal cortex, and subiculum. In addition, moderate levels of expression were observed in several hypothalamic areas involved in neuroendocrine regulation, such as the suprachiasmatic, supraoptic, paraventricular, and arcuate nuclei. Moreover, activin receptors were also expressed in regions with inputs to the hypothalamus, both in the forebrain (bed nucleus of the stria terminalis and medial preoptic area) and within the brainstem (nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus, and mesencephalic raphé system). ActRII mRNA was observed in the intermediate lobe of the pituitary and, less prominently, in the anterior lobe, whereas ActRIIB appeared to be weakly expressed throughout all three pituitary divisions. In both male and female gonads, activin receptor message was clearly present in germ cells, and ActRII was the predominant form. In the ovary, in addition to an intense signal in the oocyte, activin receptor was expressed in corpus luteum and granulosa cells during diestrous day 1. In the testis, there was a strong ActRII signal in rounded spermatids, and a moderate signal in pachytene spermatocytes. In contrast, ActRIIB was absent within tubules, but weakly expressed in interstitial and Leydig cells. This is the first report of the distribution of activin receptor message in adult mammalian tissues. Although consistent with some previously suggested functional associations of activin-containing pathways in the brain, this pattern of expression suggests a greater role for activin than was previously appreciated in cortical, limbic, and somatosensory pathways and in the maturation of germ cells in the gonads of both male and female rats.

Cloning and characterization of a transmembrane serine kinase that acts as an activin type I receptor.

Activin type II receptors are transmembrane protein-serine/threonine kinases. By using a reverse-transcription PCR assay to screen for protein kinase sequences, we isolated a cDNA clone, activin X1 receptor, from rat brain that encodes a 55-kDa transmembrane protein-serine kinase which is structurally related to other receptors in this kinase subfamily. The predicted protein consists of 509 amino acids, and the kinase domain shows 40% and 37% identity to the activin and transforming growth factor beta type II receptors, respectively. No activin-binding was observed when activin X1 receptor was expressed alone in COS-M6 cells; however, coexpression with type II activin receptors gave rise to a 68-kDa affinity-labeled complex in addition to the 85-kDa type II receptor complex. The size of this cross-linked band is consistent with the size of the type I activin receptor; furthermore, activin X1 receptor associated with type II receptors, as judged by coimmunoprecipitation with type II receptor antibodies. These data suggest that activin X1 receptor can serve as an activin type I receptor and that the diverse biological effects of activins may be mediated by a complex formed by the interaction of two transmembrane protein-serine kinases.

Characterization of type II activin receptors. Binding, processing, and phosphorylation.

Activins bind to two classes of cell surface proteins, type I receptors of approximately 50-55 kDa and type II receptors of approximately 70-75 kDa. The two cloned type II activin receptors belong to a new subfamily of transmembrane protein serine kinases. Antibodies directed against each of these cloned receptors were generated and used in immunoprecipitation experiments to study the properties of the type II receptors in vivo. Precipitation of affinity-labeled receptors, formed by chemical cross-linking of 125I-activin A, resulted in coprecipitation of type I receptor complexes; denaturation of the lysates prior to interaction with the antibodies resulted in precipitation of only type II receptors. Treatment of both affinity-labeled and metabolically labeled receptors with peptide N-glycosidase F revealed the presence of N-linked carbohydrate chains. Metabolic labeling of cells with [32P]orthophosphate indicated that both type II receptors were phosphoproteins containing predominantly phosphoserine, with small amounts of phosphothreonine, but no detectable phosphotyrosine. Analysis of tryptic phosphopeptide maps of wild-type and kinase-defective mutants suggested that at least some of the phosphorylated sites arose from autophosphorylation.

Molecular characterization of rat transforming growth factor-beta type II receptor.

A cDNA encoding the rat transforming growth factor-beta (TGF-beta) type II receptor was isolated by hybridization from a rat pituitary gland cDNA library. The rat TGF-beta type II receptor comprises 567 amino acid residues with a cysteine-rich extracellular domain, a single transmembrane domain and an intracellular protein kinase domain with predicted serine/threonine specificity. The comparison of the amino acid sequences of the rat and human TGF-beta type II receptors indicated that they are highly conserved particularly in the intracellular kinase domain. RNA blot hybridization and reverse-transcription polymerase chain reaction (RT-PCR) analyses showed that rat TGF-beta type II receptor is widely distributed in various tissues and is expressed abundantly in the ovary and lung.

Molecular and functional characterization of activin receptors.

Activins are multifunctional proteins with effects on a broad spectrum of cells and tissues. They are structurally related to a large family of growth and differentiation factors that includes the inhibins, the transforming growth factors b (TGFb), the bone morphogenetic proteins (BMP), Mullerian inhibitory substance, and a number of gene products that control the development of Drosophila and Xenopus. Although the cellular signaling mechanisms of these factors remain unclear, cDNAs encoding cell surface receptors for activin have been cloned. Those receptors are transmembrane serine kinases, suggesting a novel form of signaling. Overexpression of activin receptors in Xenopus embryos indicates that these molecules are functionally involved in the transmission of the activin signal.

Molecular cloning and binding properties of the human type II activin receptor.

A full-length cDNA for the type II human activin receptor was cloned by hybridization from a human testis cDNA library. The sequence encodes a 513 amino acid protein that is 99% identical, at the amino acid level, with the mouse type II activin receptor. The type II human activin receptor consists of an extracellular domain that specifically binds activin A with a Kd of 360 pM, a single-membrane spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity.

Cloning of a second type of activin receptor and functional characterization in Xenopus embryos.

A complementary DNA coding for a second type of activin receptor (ActRIIB) has been cloned from Xenopus laevis that fulfills the structural criteria of a transmembrane protein serine kinase. Ectodermal explants from embryos injected with activin receptor RNA show increased sensitivity to activin, as measured by the induction of muscle actin RNA. In addition, injected embryos display developmental defects characterized by inappropriate formation of dorsal mesodermal tissue. These results demonstrate that this receptor is involved in signal transduction and are consistent with the proposed role of activin in the induction and patterning of mesoderm in Xenopus embryos.

Effect of activin A on globin gene expression in purified human erythroid progenitors.

The regulatory control of human erythropoiesis through a purified protein, activin A, was examined. Previous studies using mixed populations of bone marrow cells suggested that activin A has an indirect effect on cellular proliferation and DNA synthesis of erythroid progenitors through the mediation of accessory cells. In present studies, the cultures of purified erythroid progenitors were used to examine the effect of activin A on globin gene expression. Human erythroid burst-forming units (BFU-E) were partially purified from peripheral blood, and after 8 days of culture the cells generated consisted mainly of erythroid colony-forming units (CFU-E). It was found that the subsequent 7-day cultures of these purified progenitors yielded similar numbers and size distributions of erythroid colonies, regardless of the presence of activin A in the cultures. In addition, these erythroid progenitor cells were responsive, in terms of stimulation of DNA synthesis, to the addition of erythropoietin, but not to treatment by activin A. Therefore, once the erythroid progenitors are depleted of accessory cells, activin A has little effect on both the proliferation and the DNA synthesis of these progenitors. However, when these purified erythroid progenitors were cultured in the presence of activin A, the levels of all alpha, beta, and epsilon globin transcripts and hemoglobins were significantly increased. In addition, disuccinimidyl suberate was found to chemically cross-link 125I-activin A to cell surface binding proteins (45 to 54 Kd) in both purified erythroid progenitors and K562 cells. The labeling of these binding proteins was specifically inhibited by the presence of unlabeled activin A, but not transforming growth factor-beta. These results suggest that, in addition to its indirect effect on DNA synthesis and cellular proliferation of erythroid progenitors, activin A directly affects the levels of globin mRNAs and hemoglobins in developing human erythroid cells through its specific surface binding receptor(s).