Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. Genetic evidence suggests that ephrins may transduce signals and become tyrosine phosphorylated during embryogenesis. However, the induction and functional significance of ephrin phosphorylation is not yet clear. Here, we report that when we used ectopically expressed proteins, we found that an activated fibroblast growth factor (FGF) receptor associated with and induced the phosphorylation of ephrin B1 on tyrosine. Moreover, this phosphorylation reduced the ability of overexpressed ephrin B1 to reduce cell adhesion. In addition, we identified a region in the cytoplasmic tail of ephrin B1 that is critical for interaction with the FGF receptor; we also report FGF-induced phosphorylation of ephrins in a neural tissue. This is the first demonstration of communication between the FGF receptor family and the Eph ligand family and implicates cross talk between these two cell surface molecules in regulating cell adhesion.

Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region.

The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma was not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine to phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. Our mutational analysis also indicated that tyrosines 605 and 611 are important for EphB2 kinase activity. We propose Src kinase as a downstream effector that mediates the neuron's response to Eph receptor activation.

Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand.

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a Xenopus homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of Xenopus embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.

Tyrosine phosphorylation of L1 family adhesion molecules: implication of the Eph kinase Cek5.

The L1 family comprises transmembrane cell adhesion molecules of the immunoglobulin superfamily that play an important role in neuronal migration and axon outgrowth, fasciculation, and myelination. Consistent with a crucial role in developmental processes, mutations in L1 cause severe brain malformations. Although L1 activates intracellular signaling pathways, little is known about the membrane proximal events of L1 signaling. The cytoplasmic domains of L1 family proteins contain several conserved tyrosine residues that are potential targets for receptor tyrosine kinases. Here, we report that the L1 family protein Ng-CAM is phosphorylated on tyrosine in embryonic day 13 chicken retina. This is the first demonstration of in vivo tyrosine phosphorylation of an L1-like molecule. Because chicken embryo kinase 5 (Cek5) is a receptor tyrosine kinase expressed in neuronal processes and activated in the chicken embryonic retina, we have analyzed the possible role of Cek5 in L1 phosphorylation. The rat glioblastoma cell line B28 was stably transfected with human L1. Additional transient transfection with Cek5 cDNA led to expression of Cek5 in its tyrosine-phosphorylated, activated form. Biochemical analysis revealed that L1 is phosphorylated on tyrosine in Cek5-transfected cells but not in control transfectants. Furthermore, direct phosphorylation of the L1 cytoplasmic domain by Cek5 was demonstrated in an in vitro kinase assay. Tyrosine phosphorylation may represent a novel mechanism of signal cascade initiation through L1.

Reciprocal expression of the Eph receptor Cek5 and its ligand(s) in the early retina.

Recent evidence suggests that Eph receptor tyrosine kinases and their ligands provide positional information in the developing visual system. We previously found that the Eph receptor Cek5 is more highly expressed in the ventral than dorsal chicken embryonic retina. We now report the identification of a chicken ligand for Cek5 (cCek5-L) that is 75% identical to the ligand LERK2. In situ hybridization experiments do not reveal a dorsoventral gradient of cCek5-L transcripts in the optic tectum at Embryonic Day 8, suggesting that this ligand is not involved in guiding Cek5-expressing axons in the tectum. Surprisingly, it is in the retina that high levels of cCek5-L mRNA are present. In the early retina, cCek5-L is more highly expressed in the dorsal than the ventral aspect. Similarly, a Cek5 Ig chimera labels dorsal but not ventral retina, indicating that even if several Cek5 ligands are present, their overall distribution is complementary to that of Cek5. Hence, Cek5 and cCek5-L may both contribute to define anatomical compartments within the early retina. In contrast, in the 11-day embryonic retina the distributions of Cek5 and its ligand(s) show considerable overlap, suggesting changing functions as development progresses. In dissociated cultures of dorsal or ventral retinal cells seeded on plates coated with either receptor or ligand Ig chimeras, the interaction between Cek5 and its ligand(s) or cCek5-L and its receptor(s) is sufficient to mediate cell adhesion and allows neurite outgrowth.

The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells.

Integrin-mediated adhesion is known to stimulate production of phosphatidylinositol 4,5-bisphosphate (4,5-PIP2) and increase 4,5-PIP2 hydrolysis in response to platelet-derived growth factor (PDGF). We now show that treatment of cells with lovastatin, which inhibits modification of small GTP-binding proteins, reduced PIP2 levels and decreased calcium mobilization in response to PDGF and thrombin. In cell lysates, GTP gamma S stimulated PIP 5-kinase activity, and this effect was blocked by botulinum C3 exoenzyme, suggesting that Rho was responsible. GTP-bound recombinant Rho stimulated PIP 5-kinase activity, whereas GDP-Rho was much less potent and GTP-bound Rac was ineffective. Microinjected botulinum C3 exoenzyme caused diminished calcium mobilization in response to PDGF or thrombin. Conversely, microinjection of activated Rho reversed the decrease in calcium mobilization normally seen in nonadherent cells. These data demonstrate that Rho regulates 4,5-PIP2 synthesis and, indirectly, 4,5-PIP2 hydrolysis. They also raise the possibility that PIP2 synthesis could mediate the effects of Rho on the actin cytoskeleton.

Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids.

We demonstrated recently that a fraction of the matrix (M) protein of vesicular stomatitis virus (VSV) binds tightly to cellular membranes in vivo when expressed in the absence of other VSV proteins. This membrane-associated M protein was functional in binding purified VSV nucleocapsids in vitro. Here we show that the membrane-associated M protein is largely associated with a membrane fraction having the density of plasma membranes, indicating membrane specificity in the binding. In addition, we analyzed truncated forms of M protein to identify regions responsible for membrane association and nucleocapsid binding. Truncated M protein lacking the amino-terminal basic domain still associated with cellular membranes, although not as tightly as wild-type M protein, and could not bind nucleocapsids. In contrast, deletion of the carboxy-terminal 14 amino acids did not disrupt stable membrane association or nucleocapsid interaction. These results suggest that the amino terminus of M protein either interacts directly with membranes and nucleocapsids or stabilizes a conformation that is required for M protein to mediate both of these interactions.

Membrane association of functional vesicular stomatitis virus matrix protein in vivo.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a major structural component of the virion which is generally believed to bridge between the membrane envelope and the ribonucleocapsid (RNP) core. To investigate the interaction of M protein with cellular membranes in the absence of other VSV proteins, we examined its distribution by subcellular fractionation after expression in HeLa cells. Approximately 90% of M protein, expressed without other viral proteins, was soluble, whereas the remaining 10% was tightly associated with membranes. A similar distribution in VSV-infected cells has been observed previously. Conditions known to release peripherally associated membrane proteins did not detach M protein from isolated membranes. Membrane-associated M protein was soluble in the detergent Triton X-114, whereas soluble M protein was not, suggesting a chemical or conformational difference between the two forms. Membranes containing associated M protein were able to bind RNP cores, whereas membranes lacking M protein were not. We suggest that this membrane-bound M fraction constitutes a functional subset of M protein molecules required for the attachment of RNP cores to membranes during normal virus budding.

Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis.

Conjugates of the single-chain ribosome-inactivating protein gelonin with ligands that bind to cell surface molecules vary greatly in their cytotoxicity. Conjugates that are not endocytosed after binding to cells exhibit low cytotoxicity similar to that of free gelonin, while conjugates that are endocytosed demonstrate enhanced cytotoxicity relative to free gelonin. However, the number of internalized gelonin molecules needed to intoxicate cells to the same degree has been found to be similar for all conjugates and for free gelonin. The intracellular concentration of gelonin has to be between 2,000-10,000 molecules/cells to achieve a surviving fraction of 0.37. Our studies revealed the presence of three distinct categories of cell surface molecules, those that are efficient in mediating endocytosis of immunotoxins, those that are only moderately efficient, and those that seem not to cause internalization of bound immunotoxins.