Requirement of Shp-2 tyrosine phosphatase in lymphoid and hematopoietic cell development

Shp-1 and Shp-2 are cytoplasmic phosphotyrosine phosphatases with similar structures. Mice deficient in Shp-2 die at midgestation with defects in mesodermal patterning, and a hypomorphic mutation at the Shp-1 locus results in the moth-eaten viable (me(v)) phenotype. Previously, a critical role of Shp-2 in mediating erythroid/myeloid cell development was demonstrated. By using the RAG-2-deficient blastocyst complementation, the role of Shp-2 in lymphopoiesis has been determined. Chimeric mice generated by injecting Shp-2(-/-) embryonic stem cells into Rag-2-deficient blastocysts had no detectable mature T and B cells, serum immunoglobulin M, or even Thy-1(+) and B220(+) precursor lymphocytes. Collectively, these results suggest a positive role of Shp-2 in the development of all blood cell lineages, in contrast to the negative effect of Shp-1 in this process. To determine whether Shp-1 and Shp-2 interact in hematopoiesis, Shp-2(-/-):me(v)/me(v) double-mutant embryos were generated and the hematopoietic cell development in the yolk sacs was examined. More hematopoietic stem/progenitor cells were detected in Shp-2(-/-):me(v)/me(v) embryos than in Shp-2(-/-) littermates. The partial rescue by Shp-1 deficiency of the defective hematopoiesis caused by the Shp-2 mutation suggests that Shp-1 and Shp-2 have antagonistic effects in hematopoiesis, possibly through a bidirectional modulation of the same signaling pathway(s).     

Mice harboring a defective epidermal growth factor receptor (waved-2) have an increased susceptibility to acute dextran sulfate-induced colitis

BACKGROUND: It has been reported that epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) play an important role in colonic mucosal defense and repair. Waved-2 (wa-2) mice harboring a defect EGF-R and phenotypically similar to TGF-alpha knockout mice provide a novel approach to study the role of EGF-R ligands in the maintenance and repair of colonic mucosa. METHODS: Acute colonic mucosal injury was induced by oral administration of dextran sodium sulfate (DSS: 5 g%) given for 6 days ad libitum to wa-2 homozygotes and their genetic controls (n = 10, each group), as well as to wa-2 mice with and without exogenous EGF administration. Severity of colonic injury was assessed histologically of the entire colon and graded. A crypt damage score (CDS) reflecting all three grades of mucosal pathology was calculated. Decrease in total body weight, colon length and colonic blood content was determined for all groups. RESULTS: Thirty-eight percent of the entire colonic mucosa was destroyed in wa-2 animals compared to 15% in control mice. The CDS was 16.0 +/- 1.4 and 9.6 +/- 0.8 in wa-2 and control mice, respectively. EGF application to wa-2 mice did not reduce the severity of mucosal injury (CDS: 18.9 +/- 1.7 and 19.4 +/- 2.1 in EGF and vehicle injected mice, respectively). CONCLUSIONS: The increased susceptibility of wa-2 mice to DSS demonstrates the pivotal role of EGF-R ligands such as EGF and TGF-alpha in preserving the integrity of the colonic mucosa against mucosal injury. The missing beneficial effect of exogenous EGF administration in these mice further underlines the importance of an intact ligand/EGF-R pathway.     

A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation.

The mutant mouse waved-2 (wa-2) is strikingly similar to transforming growth factor alpha-deficient mice generated by gene targeting in embryonic stem cells. We confirm that wa-2 is a point mutation (T-->G resulting in a valine-->glycine substitution at residue 743) in the gene encoding the epidermal growth factor (EGF) receptor. wa-2 fibroblastic cells lack high-affinity binding sites for EGF, and the rate of internalization of EGF is retarded. Although the tyrosine kinase activity of wa-2 EGF receptors is significantly impaired, NIH 3T3 cells lacking endogenous EGF receptors but overexpressing recombinant wa-2 EGF receptor cDNA are mitogenically responsive to EGF. While young and adult wa-2 mice are healthy and fertile, 35% of wa-2 mice born of homozygous wa-2 mothers die of malnutrition because of impaired maternal lactation.     

Mice Harboring a Defective Epidermal Growth Factor Receptor-(Waved-2) Have an Increased Susceptibility to Acute Dextran-Sulfate-induced Colitis

Background: It has been reported that epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) play an important role in colonic mucosal defense and repair. Waved-2 (wa-2) mice harboring a defect EGF-R and phenotypically similar to TGF-alpha knockout mice provide a novel approach to study the role of EGF-R ligands in the maintenance and repair of colonic mucosa. Methods: Acute colonic mucosal injury was induced by oral administration of dextran sodium sulfate (DSS: 5 g%) given for 6 days ad libitum to wa-2 homozygotes and their genetic controls (n     

Human somatic PTPN11 mutations induce hematopoietic-cell hypersensitivity to granulocyte-macrophage colony-stimulating factor

Juvenile myelomonocytic leukemia (JMML) is a lethal disease of young children characterized by hypersensitivity of hematopoietic progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF). Mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp-2, are common in JMML. We hypothesized that PTPN11 mutations induce hypersensitivity of hematopoietic progenitors to GM-CSF and confer increased GM-CSF-stimulated phospho-extracellular signal-regulated kinase (Erk) levels. To test this hypothesis, the wild-type (WT) and 3 mutant Ptpn11 cDNAs (E76K, D61V, and D61Y) were transduced into murine bone marrow cells to examine GM-CSF-stimulated granulocyte-macrophage colony-forming unit (CFU-GM) growth, macrophage progenitor proliferation, and activation of the Ras signaling pathway. Expression of the Shp-2 mutants induced progenitor cell hypersensitivity to GM-CSF compared with cells transduced with vector alone or WT Shp-2. Macrophage progenitors expressing the Shp-2 mutants displayed both basal and GM-CSF-stimulated hyperproliferation compared with cells transduced with vector alone or WT Shp-2. Consistently, macrophage progenitors transduced with the Shp-2 mutants demonstrated constitutively elevated phospho-Erk levels and sustained activation of phospho-Erk following GM-CSF stimulation compared with vector alone or WT Shp-2. These data support the hypothesis that PTPN11 mutations induce hematopoietic progenitor hypersensitivity to GM-CSF due to hyperactivation of the Ras signaling axis and provide a basis for the GM-CSF signaling pathway as a target for rational drug design in JMML.     

The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants

The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.     

Role of ERK1/2 signaling in congenital valve malformations in Noonan syndrome

Noonan syndrome (NS) is the most common nonchromosomal genetic disorder associated with cardiovascular malformations. The most prominent cardiac defects in NS are pulmonary valve stenosis and hypertrophic cardiomyopathy. Gain-of-function mutations in the protein tyrosine phosphatase Shp2 have been identified in 50% of NS families. We created a NS mouse model with selective overexpression of mutant Shp2 (Q79R-Shp2) in the developing endocardial cushions. In our model, Cre recombinase driven by the Tie2 promoter irreversibly activates transgenic Q79R-Shp2 expression in the endothelial-derived cell lineage. Q79R-Shp2 expression resulted in embryonic lethality by embryonic day 14.5. Importantly, mutant embryos showed significantly enlarged endocardial cushions in the atrioventricular canal and in the outflow tract. In contrast, overexpression of wild-type Shp2 protein at comparable levels did not enhance endocardial cushion growth or alter the morphology of the mature adult valves. Expression of Q79R-Shp2 was accompanied by increased ERK1/2 activation in a subset of cells within the cushion mesenchyme, suggesting that hyperactivation of this signaling pathway may play a pathogenic role. To test this hypothesis in vivo, Q79R-Shp2-expressing mice were crossed with mice carrying either a homozygous ERK1 or a heterozygous ERK2 deletion. Deletion of ERK1 completely rescued the endocardial cushion phenotype, whereas ERK2 protein reduction did not affect endocardial cushion size. Constitutive hyperactivation of ERK1/2 signaling alone with a transgenic approach resulted in a phenocopy of the valvular phenotype. The data demonstrate both necessity and sufficiency of increased ERK activation downstream of Shp2 in mediating abnormal valve development in a NS mouse model.     

The PTEN tumor suppressor homolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway

Inactivation of the tumor suppressor PTEN gene is found in a variety of human cancers and in cancer predisposition syndromes. Recently, PTEN protein has been shown to possess phosphatase activity on phosphatidylinositol 3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase. We have identified a homolog of PTEN in Caenorhabditis elegans and have found that it corresponds to the daf-18 gene, which had been defined by a single, phenotypically weak allele, daf-18(e1375). By analyzing an allele, daf-18(nr2037), which bears a deletion of the catalytic portion of CePTEN/DAF-18, we have shown that mutation in daf-18 can completely suppress the dauer-constitutive phenotype caused by inactivation of daf-2 or age-1, which encode an insulin receptor-like molecule and the catalytic subunit of phosphatidylinositol 3-kinase, respectively. In addition, daf-18(nr2037) dramatically shortens lifespan, both in a wild-type background and in a daf-2 mutant background that normally prolongs lifespan. The lifespan in a daf-18(nr2037) mutant can be restored to essentially that of wild type when combined with a daf-2 mutation. Our studies provide genetic evidence that, in C. elegans, the PTEN homolog DAF-18 functions as a negative regulator of the DAF-2 and AGE-1 signaling pathway, consistent with the notion that DAF-18 acts a phosphatidylinositol 3,4,5-trisphosphate phosphatase in vivo. Furthermore, our studies have uncovered a longevity-promoting activity of the PTEN homolog in C. elegans.     

A tumor suppressor role for thyroid hormone beta receptor in a mouse model of thyroid carcinogenesis

We have created a knockin mutant mouse by targeting a mutation (PV) into the thyroid hormone receptor beta gene (TRbetaPV mouse). TRbetaPV/PV mice, but not TRbetaPV/+ mice, spontaneously develop follicular thyroid carcinoma. To identify other genetic changes in the TRbeta gene that could also induce thyroid carcinoma, we crossed TRbetaPV mice with TRbeta-/- mice. As TRbetaPV/- mice (mutation of one TRbeta allele in the absence of the other wild-type allele) aged, they also spontaneously developed follicular thyroid carcinoma through the pathological progression of hyperplasia, capsular and vascular invasion, anaplasia, and eventually metastasis to the lung, but not to the lymph nodes. The pathological progression of thyroid carcinoma in TRbetaPV/- mice was indistinguishable from that in TRbetaPV/PV mice. Analyses of the expression patterns of critical genes indicated activation of the signaling pathways mediated by TSH, peptide growth factors (epidermal growth factor and fibroblast growth factor), TGF-beta, TNF-alpha, and nuclear factor-kappaB, and also suggested progressive repression of the pathways mediated by the peroxisome proliferator-activated receptor gamma. The patterns in the alteration of these signaling pathways are similar to those observed in TRbeta(PV/PV) mice during thyroid carcinogenesis. These results indicate that in the absence of a wild-type allele, the mutation of one TRbeta allele is sufficient for the mutant mice to spontaneously develop follicular thyroid carcinoma. These results provide, for the first time, in vivo evidence to suggest that the TRbeta gene could function as a tumor suppressor gene. Importantly, these findings present the possibility that TRbeta could serve as a novel therapeutic target in thyroid cancer.     

TIMP-2 disrupts FGF-2-induced downstream signaling pathways

We have previously reported that tissue inhibitor of metalloproteinases-2 (TIMP-2), an endogenous inhibitor of matrix metalloproteinase, modulates angiogenic responses through the MMP inhibition-independent activity. In this study, we investigate the molecular mechanisms of TIMP-2-mediated growth inhibition in response to fibroblast growth factor-2 (FGF-2). Pre-treatment with a protein tyrosine phosphatase inhibitor orthovanadate or expression of a dominant negative Shp-1 mutant fails to induce TIMP-2 inactivation of FGF-2 signaling pathways in human microvascular endothelial cells. We also show that TIMP-2 inhibition of FGF-2-induced p42/44(MAPK) activation and cell proliferation is associated with TIMP-2 binding to integrin alpha3beta1 on endothelial cell surfaces, as demonstrated by use of anti-integrin alpha3 or beta1 blocking antibodies, or disruption of integrin alpha3 expression by siRNA. Collectively, our results indicate that TIMP-2 inhibits FGF-2 signaling pathways through association with integrin alpha3beta1 and Shp-1-dependent inhibition of p42/44(MAPK) signaling, which in turn, results in suppression of FGF-2-stimulated endothelial cell mitogenesis.     

TIMP-2 disrupts FGF-2-induced downstream signaling pathways

We have previously reported that tissue inhibitor of metalloproteinases-2 (TIMP-2), an endogenous inhibitor of matrix metalloproteinase, modulates angiogenic responses through the MMP inhibition-independent activity. In this study, we investigate the molecular mechanisms of TIMP-2-mediated growth inhibition in response to fibroblast growth factor-2 (FGF-2). Pre-treatment with a protein tyrosine phosphatase inhibitor orthovanadate or expression of a dominant negative Shp-1 mutant fails to induce TIMP-2 inactivation of FGF-2 signaling pathways in human microvascular endothelial cells. We also show that TIMP-2 inhibition of FGF-2-induced p42/44^MAPK activation and cell proliferation is associated with TIMP-2 binding to integrin α3β1 on endothelial cell surfaces, as demonstrated by use of anti-integrin α3 or β1 blocking antibodies, or disruption of integrin α3 expression by siRNA. Collectively, our results indicate that TIMP-2 inhibits FGF-2 signaling pathways through association with integrin α3β1 and Shp-1-dependent inhibition of p42/44^MAPK signaling, which in turn, results in suppression of FGF-2-stimulated endothelial cell mitogenesis.     

Bone defects in latent TGF-beta binding protein (Ltbp)-3 null mice; a role for Ltbp in TGF-beta presentation

The latent transforming growth factor (TGF)-beta binding proteins (LTBP)-1, -3 and -4 bind the latent form of the multipotent cytokine TGF-beta. To examine the function of the LTBPs, we made a null mutation of Ltbp-3 by gene targeting. The homozygous mutant animals developed cranio-facial malformations by 12 days. By three months, there was a pronounced rounding of the cranial vault, extension of the mandible beyond the maxilla, and kyphosis. The mutant animals developed osteosclerosis of the long bones and vertebrae as well as osteoarthritis between 6 and 9 months of age. These latter phenotypic changes were similar to those described for mice that have impaired TGF-beta signaling. Thus, we suggest that Ltbp-3 plays an important role in regulating TGF-beta bioavailability as the phenotype of the Ltbp-3 null mouse appears to result from decreased TGF-beta signaling. Histological examination of the skulls from null animals revealed no effects on calvarial suture closure. However, the synchondroses in the skull base were obliterated within 2 weeks of birth. This is in contrast to the wild-type synchondroses, which remain unossified throughout the life of the animal and enable growth of the skull base through endochondral ossification. Histological changes in mutant basooccipital-basosphenoid synchondrosis were observed 1.5 days after birth. Compared with wild-type or heterozygous littermates, the basooccipital-basosphenoid synchondrosis of Ltbp-3 null mice contained increased numbers of hypertrophic chondrocytes. The expression of bone sialoprotein-1 (a marker for osteoblasts) was observed in cells surrounding the synchondrosis at postnatal day 1.5 indicating ectopic ossification. The expression of Indian hedgehog (Ihh) (a marker for chondrocytes committed to hypertrophic differentiation) was found through the basooccipital-basosphenoid synchondrosis, whereas the expression of parathyroid hormone related protein (PTHrP), which inhibits chondrocyte differentiation, appeared to be diminished in Ltbp-3 null mice. This suggests that Ltbp-3 may control chondrocyte differentiation by regulating TGF-beta availability. TGF-beta may regulate PTHrP expression either downstream of Ihh or independently of Ihh signaling.     

A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis

Homozygous mutant (Shp-2Delta46-110) embryonic stem (ES) cells exhibit decreased hematopoiesis; however, the point at which Shp-2 is critical for ES cell differentiation to hematopoietic cells is unknown. We characterized the differentiation defect of Shp-2Delta46-110 ES cells by examining early points of differentiation, conducting leukemia inhibitory factor (LIF)-stimulated biochemical analysis, and performing in vitro reconstitution studies with wild-type (WT) Shp-2. ES cell in vitro differentiation assays were used to compare the differentiation of WT, Shp-2Delta46-110, and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangioblasts, by measuring blast colony-forming cell (BL-CFC) formation and flk-1 expression, and to hematopoietic progenitor colony-forming cells, by performing secondary plating assays. LIF-stimulated phospho-Stat3 (known to be critical for ES cell self-renewal and maintenance of an undifferentiated state) and phospho-Erk levels were examined by immunoblotting. ES cell survival, using annexin V staining, and secondary embryoid body (EB) formation were also evaluated. Differentiation to both mesoderm and hemangioblasts was lower in Shp-2Delta46-110 cells compared to WT cells. On reconstitution with WT Shp-2, expression of brachyury and flk-1 and differentiation to hemangioblasts and primitive and definitive hematopoietic progenitors were restored. LIF-stimulated phospho-Stat3 levels were higher, whereas phospho-Erk levels were lower in Shp-2Delta46-110 ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shp-2Delta46-110 ES cell secondary EB formation and survival. We conclude that normal Shp-2 function is critical for the initial step of ES cell differentiation to mesoderm and to hemangioblasts and acts within the LIF-gp130-Stat3 pathway to maintain a proper balance of ES cell differentiation, pluripotency, and apoptosis.     

Double mutations in klotho and osteoprotegerin gene loci rescued osteopetrotic phenotype

Klotho gene mutant mice (klotho mice, also called kl/kl) exhibit osteopetrosis in the metaphysis of femora and tibiae and die within 3 months. We previously showed by semiquantitative RT-PCR that osteoprotegerin (opg) expression levels in klotho mice were about 2-fold higher than those in wild-type mice in the bone marrow, spleen, and lung. To examine whether the high osteoprotegerin expression levels account for the osteopetrotic phenotype in the klotho homozygous mutant mice in vivo, we made double mutant mice by crossing klotho mutant and osteoprotegerin-deficient mice. Micro computed tomography analysis in the two-dimensional sagittal planes of the metaphyses and cross-sections of femoral midshaft revealed that the abnormally high fractional trabecular bone volume in klotho homozygous mice (kl/kl; 29.71%), which was about 4-fold higher compared with that of wild-type [klotho (+/+) opg (+/+)] mice (7.81%), was rescued by the coexistence of heterozygous mutation in opg gene locus (+/-; 8.36%). Single heterozygous mutation in the opg gene locus alone (without klotho mutation) did not show phenotype (trabecular bone volume, 5.84%; not significantly different from wild type). High levels of osteoprotegerin mRNA expression in the bone marrow in klotho mutant mice were reduced by the heterozygous mutation in the opg gene locus. Furthermore, high osteoprotegerin protein levels in klotho mutant mice were also reduced by the heterozygous mutations in opg gene locus. Thus, elevated levels of osteoprotegerin in mutant mice contribute at least in part to reveal the osteopetrotic phenotype in klotho mice.     

Progression toward tumor cell phenotype is enhanced by overexpression of a mutant p53 tumor-suppressor gene isolated from nasopharyngeal carcinoma.

We recently reported the detection of a heterozygous G-->C point mutation at codon 280 of p53 in nasopharyngeal carcinoma, which causes an Arg-->Thr substitution. To test whether this mutant p53 has gained function as an oncogene, we overexpressed the mutant p53 in nontumorigenic cells of two model systems: (i) human Saos-2 cells lacking endogenous p53 and (ii) mouse JB6 variants that bear endogenous wild-type p53. Although they have no growth advantage over the neomycin controls in monolayer culture, human Saos-2 transfectants overexpressing mutant p53 do show enhanced progression to tumor cell phenotype, as assayed by anchorage-independent growth and in vivo tumorigenicity. The enhancement is seen only in transfectants expressing higher levels of p53 protein. In the mouse JB6 system, the mutant p53 functions dominantly in the presence of endogenous wild-type p53 to enhance progression of preneoplastic promotion-sensitive cells toward anchorage-independent phenotype. Mouse JB6 transfectants of mutant p53 are, however, not tumorigenic in nude mice. We conclude from these studies that the G-->C point mutation of p53 at codon 280 is a gain-of-function mutation that appears to operate dominantly and that the mutant p53-thr280 has only moderate oncogenic activity. This mutation may cooperate with other yet-to-be isolated genes in the genesis of nasopharyngeal carcinoma.     

Estrogen receptor-alpha is required by the supporting somatic cells for spermatogenesis

The gene for estrogen receptor-alpha (ERalpha) was disrupted in embryonic stem cells by homologous recombination and these cells were used to generate mice with a targeted mutation in the ERalpha gene (alphaERKO mice). It was found that males homozygous for the mutation are infertile, indicating that estrogen signaling through this nuclear hormone receptor is required for male reproductive function. Although spermatogenesis appears normal in juvenile and young adult alphaERKO mice, the sperm produced are unable to fertilize eggs in vitro. To determine whether ERalpha is required by somatic or germ cells in the male reproductive tract, we transplanted germ cells from homozygous mutant (ERalpha(-/-)) males to the testes of wild-type (ERalpha(+/+)) males depleted of germ cells by busulfan treatment. The recipients ('surrogate fathers') sired offspring heterozygous for the mutation (ERalpha(+/-)) and carrying the coat-color marker of the infertile donor males. This indicated that ERalpha(-/-) germ cells are able to produce sperm competent to fertilize when they are supported by ERalpha(+/+) somatic cells. When ERalpha(+/-) offspring produced by germ cell transplantation were mated to produce ERalpha(-/-) males, these mice were found to have the same phenotype as originally reported for alphaERKO males. These studies showed that male germ cells do not require ERalpha for regulation of their own genes for development and function, and strongly imply that somatic cells of the male reproductive tract require ERalpha to support the production of sperm that are capable of fertilization.     

Potential involvement of FRS2 in insulin signaling

Shp-2 is implicated in several tyrosine kinase receptor signaling pathways. This phosphotyrosine phosphatase is composed of a catalytic domain in its C-terminus and two SH2 domains in its N-terminus. Shp-2 becomes activated upon binding through one or both SH2 domains to tyrosine phosphorylated molecules such as Shc or insulin receptor substrates. We were interested in finding a new molecule(s), tyrosine phosphorylated by the insulin receptor (IR), that could interact with Shp-2. To do so, we screened a human placenta complementary DNA (cDNA) library with the SH2 domain-containing part of Shp-2 using a modified yeast two-hybrid system. In this system we induce or repress the expression of a constitutive active IR beta-subunit. When expressed, IR phosphorylates proteins produced from the library that can then associate with Shp-2. Using this approach, we isolated FRS2 as a potential target for tyrosine phosphorylation by the IR. After cloning the entire cDNA, we found that 1) in the yeast two-hybrid system, FRS2 interacts with Shp-2 in a fashion dependent on the presence of the IR; and 2) in the PC12/IR cell-line, insulin leads to an increase in FRS2 association with the phosphatase. We next wanted to determine whether FRS2 could be a direct substrate for IR. In an in vitro kinase assay we found that wheat-germ agglutinin-purified IR phosphorylates glutathione-S-transferase-FRS2 fusion protein. Finally, in intact cells we show that insulin stimulates tyrosine phosphorylation of endogenous FRS2. In summary, by screening a two-hybrid cDNA library, we have isolated FRS2 as a possible substrate for IR. We found that IR can directly phosphorylate FRS2. Moreover, in intact cells insulin stimulates tyrosine phosphorylation of FRS2 and its subsequent association with Shp-2. Taken together these results suggest that FRS2 could participate in insulin signaling by recruiting Shp-2 and, hence, could function as a docking molecule similar to insulin receptor substrate proteins.     

Regenerative phenotype in mice with a point mutation in transforming growth factor beta type I receptor (TGFBR1)

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N-ethyl-N-nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-β, TGFBR1. Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-β target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing.     

Different functions of HetR, a master regulator of heterocyst differentiation in Anabaena sp. PCC 7120, can be separated by mutation

The HetR protein has long been recognized as a key player in the regulation of heterocyst development. HetR is known to possess autoproteolytic and DNA-binding activities. During a search for mutants of Anabaena sp. PCC 7120 that can overcome heterocyst suppression caused by overexpression of the patS gene, which encodes a negative regulator of differentiation, a bypass mutant strain, S2-45, was isolated that produced a defective pattern (Pat phenotype) of irregularly spaced single and multiple contiguous heterocysts (Mch phenotype) in combined nitrogen-free medium. Analysis of the S2-45 mutant revealed a R223W mutation in HetR, and reconstruction in the wild-type background showed that this mutation was responsible for the Mch phenotype and resistance not only to overexpressed patS, but also to overexpressed hetN, another negative regulator of differentiation. Ectopic overexpression of the hetRR223W allele in the hetRR223W background resulted in a conditionally lethal (complete differentiation) phenotype. Analysis of the heterocyst pattern in the hetRR223W mutant revealed that heterocysts differentiate essentially randomly along filaments, indicating that this mutation results in an active protein that is insensitive to the major signals governing heterocyst pattern formation. These data provide genetic evidence that, apart from being an essential activator of differentiation, HetR plays a central role in the signaling pathway that controls the heterocyst pattern.