Direct binding of Reelin to VLDL receptor and ApoE receptor 2 induces tyrosine phosphorylation of disabled-1 and modulates tau phosphorylation.

The large extracellular matrix protein Reelin is produced by Cajal-Retzius neurons in specific regions of the developing brain, where it controls neuronal migration and positioning. Genetic evidence suggests that interpretation of the Reelin signal by migrating neurons involves two neuronal cell surface proteins, the very low density lipoprotein receptor (VLDLR) and the apoE receptor 2 (ApoER2) as well as a cytosolic adaptor protein, Disabled-1 (Dab1). We show that Reelin binds directly and specifically to the ectodomains of VLDLR and ApoER2 in vitro and that blockade of VLDLR and ApoER2 correlates with loss of Reelin-induced tyrosine phosphorylation of Disabled-1 in cultured primary embryonic neurons. Furthermore, mice that lack either Reelin or both VLDLR and ApoER2 exhibit hyperphosphorylation of the microtubule-stabilizing protein tau. Taken together, these findings suggest that Reelin acts via VLDLR and ApoER2 to regulate Disabled-1 tyrosine phosphorylation and microtubule function in neurons.

Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development.

BACKGROUND: The extracellular protein Reln controls neuronal migrations in parts of the cortex, hippocampus and cerebellum. In vivo, absence of Reln correlates with up-regulation of the docking protein Dab1 and decreased Dab1 tyrosine phosphorylation. Loss of the Reln receptor proteins, apolipoprotein receptor 2 and very low density lipoprotein receptor, results in a Reln-like phenotype accompanied by increased Dab1 protein expression. Complete loss of Dab1, however, recapitulates the Reln phenotype. RESULTS: To determine whether Dab1 tyrosine phosphorylation affects Dab1 protein expression and positioning of embryonic neurons, we have identified Dab1 tyrosine phosphorylation sites. We then generated mice in which the Dab1 protein had all the potential tyrosine phosphorylation sites mutated. This mutant protein is not tyrosine phosphorylated during brain development and is not upregulated to the extent observed in the Reln or the apoER2 and VLDLR receptor mutants. Animals expressing the non-phosphorylated Dab1 protein have a phenotype similar to the dab1-null mutant. CONCLUSIONS: Dab1 is downregulated by the Reln signal in neurons in the absence of tyrosine phosphorylation. Dab1 tyrosine phosphorylation sites and not downregulation of Dab1 protein are required for Reln signaling.

Csk suppression of Src involves movement of Csk to sites of Src activity.

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.

Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element.

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

Characterization of a cDNA clone encoding murine mitogen-regulated protein: regulation of mRNA levels in mortal and immortal cell lines.

Mitogen-regulated protein (MRP) is secreted by certain immortal murine cell lines (Swiss 3T3, BNL) stimulated with serum or particular growth factors. We have identified a cDNA clone that encodes part of the protein and have confirmed that MRP is closely related to, if not identical to, the prolactin-related protein designated proliferin. MRP is not produced by primary mouse embryo fibroblasts to nearly the same extent as it is produced by many immortal or transformed lines. Control of expression of this protein by growth factors is achieved both by regulating the extent of transcription and by regulating the processing of the protein.

Regulation of Btk by Src family tyrosine kinases.

Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed.

The disabled 1 phosphotyrosine-binding domain binds to the internalization signals of transmembrane glycoproteins and to phospholipids.

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases.

We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.

Multiple cDNAs encoding the esk kinase predict transmembrane and intracellular enzyme isoforms.

A novel protein kinase, the Esk kinase, has been isolated from an embryonal carcinoma (EC) cell line by using an expression cloning strategy. Sequence analysis of two independent cDNA clones (2.97 and 2.85 kb) suggested the presence of two Esk isoforms in EC cells. The esk-1 cDNA sequence predicted an 857-amino-acid protein kinase with a putative membrane-spanning domain, while the esk-2 cDNA predicted an 831-amino-acid kinase which lacked this domain. In adult mouse cells, esk mRNA levels were highest in tissues possessing a high proliferation rate or a sizeable stem cell compartment, suggesting that the Esk kinase may play some role in the control of cell proliferation or differentiation. As anticipated from the screening procedure, bacterial expression of the Esk kinase reacted with antiphosphotyrosine antibodies on immunoblots. Furthermore, in in vitro kinase assays, the Esk kinase was shown to phosphorylate both itself and the exogenous substrate myelin basic protein on serine, threonine, and tyrosine residues, confirming that the Esk kinase is a novel member of the serine/threonine/tyrosine family of protein kinases.

The LDL receptor gene family: signaling functions during development.

The traditional views regarding the biological functions of the low-density lipoprotein (LDL) receptor gene family have been revisited recently with new evidence that at least some of the members of this receptor family act as signal-transduction molecules. Known for their role in endocytosis, particularly of their namesake the LDLs, and for their role in the prevention of atherosclerosis, these receptors belong to an ancient family with numerous ligands, effector molecules and functions. Recent evidence implicates this family of receptors in diverse signaling pathways, long-term potentiation and neuronal degeneration.

Protein kinase C gamma mutations in spinocerebellar ataxia 14 increase kinase activity and alter membrane targeting.

The protein kinase C gamma (PKCgamma) gene is mutated in spinocerebellar ataxia type 14 (SCA14). In this study, we investigated the effects of two SCA14 missense mutations, G118D and C150F, on PKCgamma function. We found that these mutations increase the intrinsic activity of PKCgamma. Direct visualization of labelled PKCgamma in living cells demonstrates that the mutant protein translocates more rapidly to selected regions of the plasma membrane in response to Ca2+ influx. These results point to specific alterations in mutant PKCgamma function that could lead to the selective neuronal degeneration of SCA14.

Thyroid hormone regulates reelin and dab1 expression during brain development.

The reelin and dab1 genes are necessary for appropriate neuronal migration and lamination during brain development. Since these processes are controlled by thyroid hormone, we studied the effect of thyroid hormone deprivation and administration on the expression of reelin and dab1. As shown by Northern analysis, in situ hybridization, and immunohistochemistry studies, hypothyroid rats expressed decreased levels of reelin RNA and protein during the perinatal period [embryonic day 18 (E18) and postnatal day 0 (P0)]. The effect was evident in Cajal-Retzius cells of cortex layer I, as well as in layers V/VI, hippocampus, and granular neurons of the cerebellum. At later ages, however, Reelin was more abundant in the cortex, hippocampus, cerebellum, and olfactory bulb of hypothyroid rats (P5), and no differences were detected at P15. Conversely, Dab1 levels were higher at P0, and lower at P5 in hypothyroid animals. In line with these results, reelin RNA and protein levels were higher in cultured hippocampal slices from P0 control rats compared to those from hypothyroid animals. Significantly, thyroid-dependent regulation of reelin and dab1 was confirmed in vivo and in vitro by hormone treatment of hypothyroid rats and organotypic cultures, respectively. In both cases, thyroid hormone led to an increase in reelin expression. Our data suggest that the effects of thyroid hormone on neuronal migration may be in part mediated through the control of reelin and dab1 expression during brain ontogenesis.

Cerebellar abnormalities in the disabled (mdab1-1) mouse.

A mouse homolog of the Drosophila Disabled (dab) gene, disabled-1 (mdab1), encodes an adaptor molecule that functions in neural development. Targeted disruption of the mdab1 gene (mdab1-1 mice) leads to anomalies in the development of the cerebrum, hippocampus, and cerebellum. Here we describe a number of histologic abnormalities in the cerebellum of the mdab1-1 mouse. There is a complete absence of foliation, and most Purkinje cells are clumped in central clusters. However, lamination appears to develop normally in areas where the Purkinje cells and external granular layer are closely apposed. The granular layer forms a thin rind over most of the cerebellar surface, but is subdivided by both transverse and parasagittal boundaries. The Purkinje cells, identified by anti-zebrin II in the adult or anti-calbindin in the new born mdab1-1 mutant cerebellum, form a parasagittal banding pattern, similar to but distorted compared with the wild-type design. The data suggest that the development of the mdab1-1 cerebellum parallels the development of reeler. The reeler gene encodes an extracellular protein (Reelin) that is secreted by the external granular layer. Because Reelin expression is retained in the mdab1-1 mutant mouse, mDab1 p80 may act in a parallel pathway or downstream of Reelin, leading to the transformation of embryonic Purkinje cell clusters into the adult parasagittal bands.

Beta amyloid-independent role of amyloid precursor protein in generation and maintenance of dendritic spines.

Synapse loss induced by amyloid beta (Abeta) is thought to be a primary contributor to cognitive decline in Alzheimer's disease. Abeta is generated by proteolysis of amyloid precursor protein (APP), a synaptic receptor whose physiological function remains unclear. In the present study, we investigated the role of APP in dendritic spine formation, which is known to be important for learning and memory. We found that overexpression of APP increased spine number, whereas knockdown of APP reduced spine density in cultured hippocampal neurons. This spine-promoting effect of APP required both the extracellular and intracellular domains of APP, and was accompanied by specific upregulation of the GluR2, but not the GluR1, subunit of AMPA receptors. In an in vivo experiment, we found that cortical layers II/III and hippocampal CA1 pyramidal neurons in 1 year-old APP-deficient mice had fewer and shorter dendritic spines than wild-type littermates. In contrast, transgenic mice overexpressing mutant APP exhibited increased spine density compared to control animals, though only at a young age prior to overaccumulation of soluble amyloid. Additionally, increased glutamate synthesis was observed in young APP transgenic brains, whereas glutamate levels were decreased and GABA levels were increased in APP-deficient mice. These results demonstrate that APP is important for promoting spine formation and is required for proper spine development.

Mouse disabled (mDab1): a Src binding protein implicated in neuronal development.

Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development. We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks.

Reelin-induced tyrosine [corrected] phosphorylation of disabled 1 during neuronal positioning.

The reelin (reln) and disabled 1 (dab1) genes both ensure correct neuronal positioning during brain development. We have found that the intracellular Dab1 protein receives a tyrosine phosphorylation signal from extracellular Reln protein. Genetic analysis shows that reln function depends on dab1, and vice versa, as expected if both genes are in the same pathway. Dab1 is expressed at a higher level, yet phosphorylated at a lower level, in reln mutant embryo brains. In primary neuronal cultures, Dab1 tyrosine phosphorylation is stimulated by exogenous Reln. These results suggest that Reln regulates neuronal positioning by stimulating Dab1 tyrosine phosphorylation.

Deletion of the SH3 domain of Src interferes with regulation by the phosphorylated carboxyl-terminal tyrosine.

A current model for the regulation of the Src protein-tyrosine kinase proposes that the COOH-terminal phosphotyrosine, Tyr-527, binds to the Src homology 2 (SH2) region in an intramolecular interaction that represses the kinase domain. This model is consistent with the activation of Src by mutations in the SH2 domain or COOH terminus. Mutations in the SH3 domain also activate Src, although this region is not thought to bind phosphotyrosine. Seidel-Dugan et al. (Seidel-Dugan, C., Meyer, B. E., Thomas, S. M., and Brugge, J. S. (1992) Mol. Cell. Biol. 12, 1835-1845) have shown that Src mutants with deletions in the SH2 or SH3 domain transform chicken embryo fibroblasts and have increased kinase activity. These mutant proteins are underphosphorylated at Tyr-527, a change that could in itself activate the mutants. Therefore, it is not possible to distinguish whether the SH2 and SH3 domains are needed for phosphorylation of Tyr-527 or for Src to adopt or maintain the repressed state. We have artificially increased the level of Tyr-527 phosphorylation of SH2 and SH3 deletion mutants by coexpressing them with the Tyr-527 kinase, Csk, in yeast cells. We find that both the SH2 and SH3 domains are needed for inhibition of Src by Csk. The SH2 domain is needed for efficient phosphorylation by Csk, both in yeast cells and in vitro. The SH3 domain is needed for Src to be inhibited when Tyr-527 is phosphorylated by Csk. This suggests that the SH3 domain cooperates with the SH2 domain and phosphorylated Tyr-527 to inhibit the kinase domain. Dephosphorylation of SH3 domain mutants at Tyr-527 in fibroblasts could be a consequence of a failure of the proposed SH2/phosphotyrosine interaction.

Rat carbamyl-phosphate synthetase I gene. Promoter sequence and tissue-specific transcriptional regulation in vitro.

The region flanking the 5'-end of the rat gene encoding the cytoplasmic precursor of carbamyl-phosphate synthetase I, a mitochondrial matrix enzyme, has been cloned and partially characterized. S1 nuclease and primer extension analyses position the starts of transcription 138-140 nucleotides upstream of the translation initiation codon. Exon 1 contains this untranslated sequence and extends downstream to include the coding region for the pre-enzyme signal peptide (38 amino acids) plus 4 amino acids from the amino terminus of the mature protein. The 5'-flanking sequence contains typical promoter elements, including putative TATA and CAAT motifs at -21 and -82 nucleotides, respectively. In addition, several copies of consensus sequences corresponding to the H4TF-1 recognition element, GATTTC, together with the enhancer-like octamer, ATTTGCAT, are also present. Carbamyl-phosphate synthetase I is a cell-type specific enzyme, being expressed only in hepatocytes and epithelial cells of the intestinal mucosa. It is also synthesized at relatively high levels in the hepatoma cell line, Hep G2. Employing pCPS2.1, a minigene containing the promoter and part of exon 1, we show that nuclear extracts from Hep G2 support accurate carbamyl-phosphate synthetase I gene transcription in vitro. No such activity was observed, however, in extracts from HeLa, a cell line which does not express carbamyl-phosphate synthetase I.

Neuronal position in the developing brain is regulated by mouse disabled-1.

During mammalian brain development, immature neurons migrate radially from the neuroectoderm to defined locations, giving rise to characteristic cell layers. Here we show that targeted disruption of the mouse disabled1 (mdab1) gene disturbs neuronal layering in the cerebral cortex, hippocampus and cerebellum. The gene encodes a cytoplasmic protein, mDab1 p80, which is expressed and tyrosine-phosphorylated in the developing nervous system. It is likely to be an adaptor protein, docking to others through its phosphotyrosine residues and protein-interacting domain. The mdab1 mutant phenotype is very similar to that of the reeler mouse. The product of the reeler gene, Reelin, is a secreted protein that has been proposed to act as an extracellular signpost for migrating neurons. Because mDab1 is expressed in wild-type cortical neurons, and Reelin expression is normal in mdab1 mutants, mDab1 may be part of a Reelin-regulated or parallel pathway that controls the final positioning of neurons.

Alternative splicing of STY, a nuclear dual specificity kinase.

The LAMMER subfamily of kinases has been conserved throughout evolution, and its members are thought to play important roles in the regulation of cellular growth and differentiation programs. STY is a murine LAMMER kinase which has been implicated in the control of PC12 cell differentiation. Multiple transcripts are derived from the Sty gene, and their relative abundance is developmentally regulated. Alternative splicing of the primary Sty transcript generates mRNAs encoding full-length catalytically active (STY) and truncated, kinase-deficient polypeptides. Both STY and its truncated isoform, STYT, are localized in the nucleus and are capable of heterodimerizing. We also demonstrate that STY functions as a dual specificity kinase in mammalian cells.