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The ROSAβ geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral β geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5′ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.

Heterozygous deletions within human chromosome 22q11 are the genetic basis of DiGeorge/velocardiofacial syndrome (DGS/VCFS), the most common deletion syndrome (1 in 4,000 live births) in humans 1 . CRKL maps within the common deletion region for DGS/VCFS (ref. 2 ) and encodes an SH2-SH3-SH3 adapter protein closely related to the Crk gene products 3 . Here we report that mice homozygous for a targeted null mutation at the CrkL locus (gene symbol Crkol for mice) exhibit defects in multiple cranial and cardiac neural crest derivatives including the cranial ganglia, aortic arch arteries, cardiac outflow tract, thymus, parathyroid glands and craniofacial structures. We show that the migration and early expansion of neural crest cells is unaffected in Crkol −/− embryos. These results therefore indicate an essential stage- and tissue-specific role for Crkol in the function, differentiation, and/or survival of neural crest cells during development. The similarity between the Crkol −/− phenotype and the clinical manifestations of DGS/VCFS implicate defects in CRKL-mediated signaling pathways as part of the molecular mechanism underlying this syndrome.

CRK and CRKL ( CRK-like ) 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 22q11.21 genes CRKL and TBX1 , Crk and Tbx1 also genetically interact, thus suggesting that pathways shared by the three genes participate in organogenesis affected in the syndrome. We also show that Crk and Crkl are required during mesoderm development, and Crk/Crkl deficiency results in small cell size and abnormal mesenchyme behavior in primary embryonic fibroblasts. Our systems-wide analyses reveal impaired glycolysis, associated with low Hif1a protein levels as well as reduced histone H3K27 acetylation in several key glycolysis genes. Furthermore, Crk/Crkl deficiency sensitizes MEFs to 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to induce cell blebbing. Activated Rapgef1, a Crk/Crkl-downstream effector, rescues several aspects of the cell phenotype, including proliferation, cell size, focal adhesions, and phosphorylation of p70 S6k1 and ribosomal protein S6. Our investigations demonstrate that Crk/Crkl-shared pathways orchestrate metabolic homeostasis and cell behavior through widespread epigenetic controls.

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. We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

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. Among del22q11.2 patients exhibiting GU anomalies, we have mapped the smallest relevant region to only five genes, including CRKL. CRKL encodes a srchomology adaptor protein implicated in mediating tyrosine kinase signaling, and is expressed in the developing GU-tract in mice and humans. Here we show that Crkl mutant embryos exhibit gene dosage-dependent growth restriction, and homozygous mutants exhibit upper GU defects at a microdissection-detectable rate of 23%. RNA-sequencing revealed that 52 genes are differentially regulated in response to uncoupling Crkl from its signaling pathways in the developing kidney, including a fivefold up-regulation of Foxd1, a known regulator of nephron progenitor differentiation. Additionally, Crkl heterozygous adult males exhibit cryptorchidism, lower testis weight, lower sperm count, and subfertility. Together, these data indicate that CRKL is intimately involved in normal development of both the upper and lower GU tracts, and disruption of CRKL contributes to the high incidence of GU defects associated with deletion at 22q11.2.

A third of patients with the DiGeorge syndrome have congenital kidney and urinary tract anomalies. This study provides evidence that haploinsufficiency of CRKL is associated with such anomalies in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Deletions on chromosome 22q11.2 are the most common cause of the DiGeorge syndrome (Online Mendelian Inheritance in Man [OMIM] number, 188400) and the velocardiofacial syndrome (OMIM number, 192430) and constitute the most common microdeletion disorder in humans, with an estimated prevalence of 1 in 2000 to 4000 live births. 1 – 3 The DiGeorge syndrome is a debilitating, multisystemic condition that features (with variable expressivity) cardiac malformations, velopharyngeal insufficiency, hypoparathyroidism with hypocalcemia, and thymic aplasia with immune deficiency. Additional phenotypes include neurodevelopmental defects and urogenital malformations. 4 – 7 The long arm of chromosome 22 contains multiple segmental duplications (low-copy repeats) that confer a . . .

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. We have isolated two orthologs expressed in the choanoflagellate Monosiga brevicollis , a unicellular relative to the metazoans. Consistent with its highly-conserved three-dimensional structure, the SH2 domain of M. brevicollis crka1 can bind to the mammalian CRK/CRKL SH2 binding consensus phospho-YxxP, and to the SRC substrate/focal adhesion protein BCAR1 (p130 CAS ) in the presence of activated SRC. These results demonstrate an ancient origin of the CRK/CRKL SH2-target recognition specificity. Although BCAR1 orthologs exist only in metazoans as identified by an N-terminal SH3 domain, YxxP motifs, and a C-terminal FAT-like domain, some pre-metazoan transmembrane proteins include several YxxP repeats in their cytosolic region, suggesting that they are remotely related to the BCAR1 substrate domain. Since the tyrosine kinase SRC also has a pre-metazoan origin, co-option of BCAR1-related sequences may have rewired the crka-dependent network to mediate adhesion signals in the metazoan ancestor.

MOUSE embryos lacking Csk, a negative regulator of Src family kinases, exhibit defects in neurulation and die at mid-gestation 1,2 . To determine the role of activated Src family kinases in the csk - phenotype, we have introduced mutations in the src and fyn genes 3,4 into the csk - mutant background. Genetic analysis reveals that src , but not fyn , is partly epistatic to the csk gene. Biochemical analysis indicates that several cytoskeletal proteins are hyperphosphorylated on tyrosine residues in csk - cells. Regulation of cortactin and tensin hyperphosphorylation is Src-dependent, whereas focal adhesion kinase and paxillin hyperphosphorylation is partly dependent on both Src and Fyn. Furthermore, the src- mutation can restore the normal distribution of cortactin and partly correct filamentous actin organization in csk - cells. Thus, Src family kinases have both specific and overlapping functions in regulation of the cytoskeleton. The disturbance of these functions may be a molecular basis for the phenotype exhibited by csk - mutants.

Enzymes are central to life, with their catalytic activity often shaped by the dynamic conformations of regulatory loops. In hub enzymes such as tyrosine kinases, the activation loop critically controls substrate specificity, catalytic efficiency, and downstream signaling, shaping cellular fate. Yet, the molecular mechanisms by which loop dynamics encode these functions remain incompletely understood. Here, we used SRC kinase as a model to dissect how minimal perturbations of the activation loop reprogram kinase behavior. By generating and characterizing multiple variants, we identified a triple-deletion mutant with altered loop dynamics. Structural and biochemical analyses revealed that this variant explores distinct loop conformations and exhibits a subtle shift in substrate preference toward more acidic motifs. These fine-tuned conformational changes translated into specific cellular signaling outcomes, as demonstrated by phosphoproteomic profiling. Comparative analysis across species further showed that nature exploits similar loop remodeling strategies to modulate kinase function. Together, our findings provide a blueprint for rationally tuning kinase activity and offer a generalizable framework for rewiring signaling pathways in diverse cellular contexts.