A hypomorphic allele of dab1 reveals regional differences in reelin-Dab1 signaling during brain development

Development ◽  
2002 ◽  
Vol 129 (3) ◽  
pp. 787-796
Author(s):  
Tara M. Herrick ◽  
Jonathan A. Cooper
Keyword(s):  
Brain Development ◽  
Tyrosine Phosphorylation ◽  
Marginal Zone ◽  
Normal Development ◽  
Single Copy ◽  
Terminal Region ◽  
Cortical Plate ◽  
Phosphorylation Sites ◽  
Ca1 Region ◽  
Reelin Signaling

The disabled 1 (Dab1) p80 protein is essential for reelin signaling during brain development. p80 has an N-terminal domain for association with reelin receptors, followed by reelin-dependent tyrosine phosphorylation sites and about 310 C-terminal residues of unknown function. We have generated mutant mice that express only a natural splice form of Dab1, p45, that lacks the C-terminal region of p80. The normal development of these mice implies that the receptor-binding region and tyrosine phosphorylation sites of p80 are sufficient for reelin signaling. However, a single copy of the truncated gene does not support normal development of the neocortex and hippocampus. The CA1 region of the hippocampus is split into two well-organized layers, while the marginal zone of the neocortex is invaded by late-born cortical plate neurons. The haploinsufficiency of the p45 allele of Dab1 implies that the C terminus of p80 affects the strength of reelin-Dab1 signaling, yet there is no apparent change in reelin-dependent tyrosine phosphorylation of p45 relative to p80. Therefore, we suggest that the C-terminal region of Dab1 p80 is involved in signaling to downstream effector molecules. Furthermore, the presence of late-born cortical plate neurons in the marginal zone reveals a requirement for reelin-Dab1 signaling in late-born cortical plate neurons, and helps distinguish models for the cortical inversion in the reeler mutant mouse.

scholarly journals Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development

2000 ◽  
Vol 10 (15) ◽  
pp. 877-885 ◽  
Author(s):  
Brian W. Howell ◽  
Tara M. Herrick ◽  
Jeffrey D. Hildebrand ◽  
Yanni Zhang ◽  
Jonathan A. Cooper
Keyword(s):  
Brain Development ◽  
Tyrosine Phosphorylation ◽  
Mouse Brain ◽  
Phosphorylation Sites ◽  
Mouse Brain Development

Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation

2012 ◽  
Vol 90 (3) ◽  
pp. 361-369 ◽  
Author(s):  
Serene Keilani ◽  
DeLacy Healey ◽  
Kiminobu Sugaya
Keyword(s):  
Brain Development ◽  
Tyrosine Phosphorylation ◽  
Cell Fate ◽  
Molecular Mechanisms ◽  
Neural Progenitor Cells ◽  
Lipid Binding ◽  
Notch 1 ◽  
Radial Glial Cells ◽  
Human Neural Progenitor Cells ◽  
Reelin Signaling

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.

scholarly journals Dual Functions of Dab1 during Brain Development

2008 ◽  
Vol 29 (2) ◽  
pp. 324-332 ◽  
Author(s):  
Libing Feng ◽  
Jonathan A. Cooper
Keyword(s):  
Brain Development ◽  
Binding Sites ◽  
Tyrosine Kinases ◽  
Normal Development ◽  
The Other ◽  
Normal Brain ◽  
Phosphorylation Sites ◽  
Src Family Tyrosine Kinases ◽  
Normal Brain Development

ABSTRACT Reelin coordinates the movements of neurons during brain development by signaling through the Dab1 adaptor and Src family tyrosine kinases. Experiments with cultured neurons have shown that when Dab1 is phosphorylated on tyrosine, it activates Akt and provides a scaffold for assembling signaling complexes, including the paralogous Crk and CrkL adaptors. The roles of Akt and Dab1 complexes during development have been unclear. We have generated two Dab1 alleles, each lacking two out of the four putative tyrosine phosphorylation sites. Neither allele supports normal brain development, but each allele complements the other. Two tyrosines are required for Reelin to stimulate Dab1 phosphorylation at the other sites, to activate Akt, and to downregulate Dab1 levels. The other two tyrosines are required to stimulate a Crk/CrkL-C3G pathway. The absence of Crk/CrkL binding sites and C3G activation causes an unusual layering phenotype. These results show that Reelin-induced Akt stimulation and Dab1 turnover are not sufficient for normal development and suggest that Dab1 acts both as a kinase switch and as a scaffold for assembling signaling complexes in vivo.

scholarly journals Maternal Ethanol Exposure Acutely Elevates Src Family Kinase Activity in the Fetal Cortex

2021 ◽  
Author(s):  
Dandan Wang ◽  
Brian W. Howell ◽  
Eric C. Olson
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cortical Neurons ◽  
Molecular Mechanisms ◽  
Fetal Brain ◽  
Ethanol Exposure ◽  
Src Family Kinase ◽  
Cortical Cells ◽  
Signaling Systems ◽  
Sustained Reduction ◽  
Reelin Signaling

AbstractFetal alcohol syndrome (FAS) is characterized by disrupted fetal brain development and postnatal cognitive impairment. The targets of alcohol are diverse, and it is not clear whether there are common underlying molecular mechanisms producing these disruptions. Prior work established that acute ethanol exposure causes a transient increase in tyrosine phosphorylation of multiple proteins in cultured embryonic cortical cells. In this study, we show that a similar tyrosine phosphorylation transient occurs in the fetal brain after maternal dosing with ethanol. Using phospho-specific antibodies and immunohistochemistry, we mapped regions of highest tyrosine phosphorylation in the fetal cerebral cortex and found that areas of dendritic and axonal growth showed elevated tyrosine phosphorylation 10 min after maternal ethanol exposure. These were also areas of Src expression and Src family kinase (SFK) activation loop phosphorylation (pY416) expression. Importantly, maternal pretreatment with the SFK inhibitor dasatinib completely prevents both the pY416 increase and the tyrosine phosphorylation response. The phosphorylation response was observed in the perisomatic region and neurites of immature migrating and differentiating primary neurons. Importantly, the initial phosphotyrosine transient (~ 30 min) targets both Src and Dab1, two critical elements in Reelin signaling, a pathway required for normal cortical development. This initial phosphorylation response is followed by sustained reduction in Ser3 phosphorylation of n-cofilin, a critical actin severing protein and an identified downstream effector of Reelin signaling. This biochemical disruption is associated with sustained reduction of F-actin content and disrupted Golgi apparatus morphology in developing cortical neurons. The finding outlines a model in which the initial activation of SFKs by ethanol has the potential to disrupt multiple developmentally important signaling systems for several hours after maternal exposure.

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

2006 ◽  
Vol 290 (2) ◽  
pp. C463-C471 ◽  
Author(s):  
Shutang Zhou ◽  
Bradley A. Webb ◽  
Robert Eves ◽  
Alan S. Mak
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tyrosine Phosphorylation ◽  
Phorbol Ester ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Muscle Cells ◽  
Phosphorylation Sites ◽  
A7r5 Cells ◽  
Sirna Knockdown

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKCα inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKCα acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation.

Structural differences between repressed and derepressed forms of p60c-src

1989 ◽  
Vol 9 (6) ◽  
pp. 2648-2656
Author(s):  
A MacAuley ◽  
J A Cooper
Keyword(s):  
Kinase Activity ◽  
Kinase Domain ◽  
Terminal Region ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Kinase Activation ◽  
Amino Terminal ◽  
Structural Differences ◽  
Carboxy Terminal ◽  
Pronase E

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively

1993 ◽  
Vol 13 (1) ◽  
pp. 133-143
Author(s):  
M Valius ◽  
C Bazenet ◽  
A Kazlauskas
Keyword(s):  
Dna Synthesis ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphates ◽  
Platelet Derived Growth Factor ◽  
Phosphorylation Sites ◽  
Carboxy Terminus ◽  
Mitogenic Response ◽  
Stable Association ◽  
64 Kda Protein ◽  
Carboxy Terminal

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates

1984 ◽  
Vol 4 (7) ◽  
pp. 1213-1220
Author(s):  
M S Collett ◽  
S K Belzer ◽  
A F Purchio
Keyword(s):  
Tyrosine Phosphorylation ◽  
Rous Sarcoma Virus ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Terminal Region ◽  
Transformed Cell ◽  
Cell Lysates ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

scholarly journals Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5

2001 ◽  
Vol 13 (2) ◽  
pp. 149-156 ◽  
Author(s):  
Kevin M. Dennehy ◽  
William F. Ferris ◽  
Hanne Veenstra ◽  
Linda A. Zuckerman ◽  
Nigel Killeen ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Phosphorylation Sites ◽  
Transmembrane Glycoprotein
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