Cell shape-based chemical screening reveals an epigenetic network mediated by focal adhesions.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators.

Essential role of the family-dosage in DiGeorge-like anomaly and metabolic homeostasis.

and () encode adapter proteins with similar biochemical properties. Here, we show that a 50% reduction of the family-combined dosage generates developmental defects, including aspects of DiGeorge/del22q11 syndrome in mice. Like the mouse homologs of two 22q11.

Murine model indicates 22q11.2 signaling adaptor is a dosage-sensitive regulator of genitourinary development.

The spectrum of congenital anomalies affecting either the upper tract (kidneys and ureters) or lower tract (reproductive organs) of the genitourinary (GU) system are fundamentally linked by the developmental origin of multiple GU tissues, including the kidneys, gonads, and reproductive ductal systems: the intermediate mesoderm. Although ∼31% of DiGeorge/del22q11.2 syndrome patients exhibit GU defects, little focus has been placed on the molecular etiology of GU defects in this syndrome.

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome.

Background: The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown.

A pre-metazoan origin of the CRK gene family and co-opted signaling network.

CRK and CRKL adapter proteins play essential roles in development and cancer through their SRC homology 2 and 3 (SH2 and SH3) domains. To gain insight into the origin of their shared functions, we have investigated their evolutionary history. We propose a term, crk/crkl ancestral (crka), for orthologs in invertebrates before the divergence of CRK and CRKL in the vertebrate ancestor.

RKIP regulates MAP kinase signaling in cells with defective B-Raf activity.

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism.

A specific need for CRKL in p210BCR-ABL-induced transformation of mouse hematopoietic progenitors.

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL), the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here, we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells.

Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

Background: Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function.

Methods/findings: We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function.

Structural and functional basis of a role for CRKL in a fibroblast growth factor 8-induced feed-forward loop.

The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics.

Raf kinase inhibitory protein (RKIP): a physiological regulator and future therapeutic target.

Background: Raf kinase inhibitory protein (RKIP) belongs to the phosphatidylethanolamine binding protein (PEBP) family that is expressed in both prokaryotic and euakaryotic organisms.

Objective: In this review, we discuss the role of RKIP as a modulator of signal transduction, the relationship of RKIP to other members of the PEBP family, and the role of RKIP in human health and disease.

Results/conclusion: In mammals, RKIP regulates activation of MAPK, NF-kappaB and G protein coupled receptors (GPCRs).

COOH-terminal Src kinase-mediated c-Jun phosphorylation promotes c-Jun degradation and inhibits cell transformation.

The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun.

Identification of the nonreceptor tyrosine kinase MATK/CHK as an essential regulator of immune cells using Matk/CHK-deficient mice.

Matk/CHK knockout mice were reported to show no apparent phenotypic abnormalities. This was thought to be due to the homologous kinase Csk that compensates for Matk/CHK. Here, we present the first evidence that the nonreceptor tyrosine kinase, Matk/CHK, is an important modulator of immune cell signaling.

Dose-dependent interaction of Tbx1 and Crkl and locally aberrant RA signaling in a model of del22q11 syndrome.

22q11 deletion (del22q11) syndrome is characterized genetically by heterozygous deletions within chromosome 22q11 and clinically by a constellation of congenital malformations of the aortic arch, heart, thymus, and parathyroid glands described as DiGeorge syndrome (DGS). Here, we report that compound heterozygosity of mouse homologs of two 22q11 genes, CRKL and TBX1, results in a striking increase in the penetrance and expressivity of a DGS-like phenotype compared to heterozygosity at either locus. Furthermore, we show that these two genes have critical dose-dependent functions in pharyngeal segmentation, patterning of the pharyngeal apparatus along the anteroposterior axis, and local regulation of retinoic acid (RA) metabolism and signaling.

Crkl deficiency disrupts Fgf8 signaling in a mouse model of 22q11 deletion syndromes.

Deletions on chromosome 22q11.21 disrupt pharyngeal and cardiac development and cause DiGeorge and related human syndromes. CRKL (CRK-Like) lies within 22q11.

Activation of a Dab1/CrkL/C3G/Rap1 pathway in Reelin-stimulated neurons.

During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype.

Dual roles of the C-terminal Src kinase (Csk) during developmental vascularization.

Here we report that C-terminal Src kinase (Csk), a tyrosine kinase that negatively regulates the activity of Src and related kinases, is important for vascular development. In Csk(-/-) embryos, although vascular tubules were formed and organized into capillary-like networks during the initial genesis of blood vessels, the vessels failed to engage in normal sprout formation. In chimeric embryos containing both wild-type and Csk(-/-) cells, the presence of wild-type cells enabled Csk(-/-) endothelial cells to participate in branching morphogenesis.

T cell development and function in CrkL-deficient mice.

The adapter protein CrkL has been implicated in multiple signal transduction pathways in hematopoietic cells. In T lymphocytes, the recruitment of CrkL-C3G complexes has been correlated with hyporesponsiveness, implicating CrkL as a potential negative regulator. To test this hypothesis we examined T cell activation in CrkL-deficient mice.

Translocation of CrkL to focal adhesions mediates integrin-induced migration downstream of Src family kinases.

The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas.

The CrkL adapter protein is required for type I interferon-dependent gene transcription and activation of the small G-protein Rap1.

We sought to determine the functional role of the CrkL adapter protein and downstream pathways in interferon signaling. In experiments using CrkL(--) mouse embryonic fibroblasts, we found that CrkL is required for IFN alpha-dependent gene transcription via GAS elements, apparently via the formation of DNA-binding complexes with Stat5. On the other hand, gene transcription via ISRE elements is intact in the absence of CrkL, indicating that the regulatory effects on gene transcription are mediated only via the formation of CrkL:Stat5 complexes.

Focal adhesions require catalytic activity of Src family kinases to mediate integrin-matrix adhesion.

Members of the Src family of tyrosine kinases function to phosphorylate focal adhesion (FA) proteins. To explore the overlapping functions of Src kinases, we have targeted Csk, a negative regulator of the Src family, to FA structures. Expression of FA-targeted Csk (FA-Csk) effectively reduced the active form (nonphosphorylated at the C-terminal regulatory tyrosine) of Src members in the cell.

The adaptor protein paxillin is essential for normal development in the mouse and is a critical transducer of fibronectin signaling.

The integrin family of cell adhesion receptors are important for a diverse set of biological responses during development. Although many integrins have been shown to engage a similar set of cytoplasmic effector proteins in vitro, the importance of these proteins in the biological events mediated by different integrin receptors and ligands is uncertain. We have examined the role of one of the best-characterized integrin effectors, the focal adhesion protein paxillin, by disruption of the paxillin gene in mice.