Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis.

BACKGROUND: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. RESULTS: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. CONCLUSIONS: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development.

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.

The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons.

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.

Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2.

The SH2 domain-containing tyrosine phosphatase Shp2 plays a pivotal role during the gastrulation of vertebrate embryos. However, because of the complex phenotype observed in mouse mutant embryos, the precise role of Shp2 during development is unclear. To define the specific functions of this phosphatase, Shp2 homozygous mutant embryonic stem cells bearing the Rosa-26 LacZ transgene were isolated and used to perform a chimeric analysis. Here, we show that Shp2 mutant cells amass in the tail bud of embryonic day 10.5 chimeric mouse embryos and that this accumulation begins at the onset of gastrulation. At this early stage, Shp2 mutant cells collect in the primitive streak of the epiblast and thus show deficiencies in their contribution to the mesoderm lineage. In high-contribution chimeras, we show that overaccumulation of Shp2 mutant cells at the posterior end of the embryo results in two abnormal phenotypes: spina bifida and secondary neural tubes. Consistent with a failure to undergo morphogenic movements at gastrulation, Shp2 is required for embryo fibroblast cells to mount a positive chemotactic response to acidic fibroblast growth factor in vitro. Our results demonstrate that Shp2 is required at the initial steps of gastrulation, as nascent mesodermal cells form and migrate away from the primitive streak. The aberrant behavior of Shp2 mutant cells at gastrulation may result from their inability to properly respond to signals initiated by fibroblast growth factors.

Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2.

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.

Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation.

Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, we have introduced a null mutation into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2-Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer.

Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2.

Shp-1, Shp-2 and corkscrew comprise a small family of cytoplasmic tyrosine phosphatases that possess two tandem SH2 domains. To investigate the biological functions of Shp-2, a targeted mutation has been introduced into the murine Shp-2 gene, which results in an internal deletion of residues 46-110 in the N-terminal SH2 domain. Shp-2 is required for embryonic development, as mice homozygous for the mutant allele die in utero at mid-gestation. The Shp-2 mutant embryos fail to gastrulate properly as evidenced by defects in the node, notochord and posterior elongation. Biochemical analysis of mutant cells indicates that Shp-2 can function as either a positive or negative regulator of MAP kinase activation, depending on the specific receptor pathway stimulated. In particular, Shp-2 is required for full and sustained activation of the MAP kinase pathway following stimulation with fibroblast growth factor (FGF), raising the possibility that the phenotype of Shp-2 mutant embryos results from a defect in FGF-receptor signalling. Thus, Shp-2 modulates tyrosine kinase signalling in vivo and is crucial for gastrulation during mammalian development.

Nuk controls pathfinding of commissural axons in the mammalian central nervous system.

Eph family receptor tyrosine kinases have been proposed to control axon guidance and fasciculation. To address the biological functions of the Eph family member Nuk, two mutations in the mouse germline have been generated: a protein null allele (Nuk1) and an allele that encodes a Nuk-beta gal fusion receptor lacking the tyrosine kinase and C-terminal domains (Nuk(lacZ)). In Nuk1 homozygous brains, the majority of axons forming the posterior tract of the anterior commissure migrate aberrantly to the floor of the brain, resulting in a failure of cortical neurons to link the two temporal lobes. These results indicate that Nuk, a receptor that binds transmembrane ligands, plays a critical and unique role in the pathfinding of specific axons in the mammalian central nervous system.

Activation and serine phosphorylation of the p56lck protein tyrosine kinase in response to antigen receptor cross-linking in B lymphocytes.

We show that cross-linking the B cell AgR with anti-Ig Abs activates p56lck (Lck) in both the immature B cell line WEHI-231 and mature resting B cells from mouse spleen. Anti-Ig-stimulated Lck activity peaked after 1 to 2 min, but remained elevated for at least 15 min. Consistent with the proposed role for src family tyrosine kinases in AgR signaling, we found that Lck could phosphorylate the cytoplasmic tails of the Ig-alpha and Ig-beta components of the B cell AgR in vitro. Lck phosphorylated both of the tyrosines in the Ig-beta AgR homology motif and one of the two tyrosines in the Ig-alpha AgR homology motif. Finally, we show that AgR ligation in B cells caused a significant portion of the Lck to migrate with an apparent molecular mass of 60 kDa on SDS-PAGE gels. Conversion of p56lck to p60lck was maximal at 5 to 15 min, at which times Lck activity in the cells was decreasing. This Lck "band shift" has been observed previously in activated T cells and correlates with phosphorylation of Lck at serine 59. We show that the 60-kDa form of Lck induced by AgR cross-linking in B cells is also phosphorylated at serine 59. Phosphorylation of Lck at this site in vitro decreases its activity. Thus, in B cells, AgR cross-linking activates Lck and subsequently activates a kinase that phosphorylates Lck at serine 59, a potential negative regulatory site.

B cell antigen receptor cross-linking induces phosphorylation of the p21ras oncoprotein activators SHC and mSOS1 as well as assembly of complexes containing SHC, GRB-2, mSOS1, and a 145-kDa tyrosine-phosphorylated protein.

Ligation of the B cell AgR activates p21ras (Ras). We have investigated the effects of AgR ligation on three proteins that have been implicated as regulators of Ras: SHC, GRB-2, and mSOS1. We show that AgR cross-linking in B cells stimulated tyrosine and serine phosphorylation of SHC. This correlated with the formation of complexes containing SHC, GRB-2, mSOS1, and an unidentified 145-kDa tyrosine-phosphorylated protein. These complexes were present in the cytosol, as well as in the membrane fraction of the cells, where Ras is located. By using a GRB-2 fusion protein to probe blots, we showed that SHC was the major protein that GRB-2 bound to in anti-Ig-stimulated B cells. This argues that SHC couples GRB-2/mSOS1 to the 145-kDa protein and that SHC is likely to be essential for mSOS1 function in B cells. Finally, we found that AgR cross-linking stimulated phosphorylation of mSOS1 and that this could be blocked by an inhibitor of protein kinase C. Thus, signaling by the B cell AgR stimulates phosphorylation of SHC and mSOS1 and induces the formation of membrane-associated complexes containing SHC, GRB-2, mSOS1, and a 145-kDa protein. These events may be important for activation of Ras by the AgR.

Evidence for a functional subclass of the RTVL-H family of human endogenous retrovirus-like sequences.

We report the discovery of a subgroup of RTVL-H human endogenous retroviral elements, designated RTVL-Hp, that is intact in the pol region which is deleted in the vast majority of RTVL-H elements. As a consequence, RTVL-Hp elements contain critical functional domains in their pol region that other RTVL-H elements lack. We estimate that the haploid genomes of humans, apes, and Old World monkeys contain 50 to 100 copies of RTVL-Hp elements and 800 to 1,000 deleted sequences. The major amplification of deleted elements appears to have occurred after the divergence of Old World and New World monkeys, since we have obtained evidence that a few intact RTVL-Hp elements, but no deleted forms, are present in marmoset DNA. Using the polymerase chain reaction coupled with a direct screen for open reading frames, we have isolated fragments from four RTVL-Hp elements amplified from human DNA that contain an open reading frame throughout a region of pol that is disrupted by diagnostic mutations in all other RTVL-H sequences that we had previously analyzed. Northern (RNA) hybridization analysis shows that unit-length RTVL-Hp transcripts are expressed in the human teratocarcinoma cell line Tera-1. Together, the results presented here suggest that a small functional subfamily of RTVL-H elements is present in the human genome.