Reelin controls neuronal positioning in the developing brain by binding to the two lipoprotein receptors, very-low-density lipoprotein receptor and apolipoprotein E receptor 2, to stimulate phosphorylation of Disabled-1 (Dab1) by the Fyn and Src tyrosine kinases. Crk and Crk-like (CrkL) have been proposed to interact with tyrosine phosphorylated Dab1 to mediate downstream events in the Reelin pathway. However, these adaptor proteins are widely expressed, and they fulfill essential functions during embryonic development. To address their specific roles in Reelin-mediated neuronal migration, we generated mutant mice, by Cre-loxP recombination, lacking Crk and CrkL in most neurons. These animals displayed the major anatomic features of reeler including, cerebellar hypofoliation, failure of Purkinje cell migration, absence of preplate splitting, impaired dendritic development, and disruption of layer formation in the hippocampus and cerebral cortex. However, proximal signaling involving tyrosine phosphorylation and turnover of Dab1 occurred normally in the mutant mouse brain and in primary cortical neurons treated with Reelin. In contrast, two downstream signaling events, Reelin-induced phosphorylation of C3G and Akt, were not observed in the absence of Crk and CrkL in mouse embryonic cortical neurons. These findings place C3G and Akt phosphorylation downstream of Crk and CrkL, which play essential overlapping functions in the Reelin signaling pathway.

The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation -markers of focal adhesion complex-mediated Crk-dependent signaling -was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2-dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases. IntroductionWhen functioning properly in health, the kidney filtration barrier selectively prevents the passage of macromolecules from the blood compartment into the urinary space. Differentiated podocytes form a remarkable octopus-like morphology, extending numerous interdigitating foot processes defined by a unique 3-dimensional actin cytoskeletal architecture and requiring formation of a specialized intercellular junction. These foot processes adhere to and cover an extracellular matrix interposed between podocytes and an endothelium that creates the glomerular capillary wall. Podocytes undergo cytoskeletal remodeling to alter their morphology in nearly all forms of human glomerular disease, exhibiting what has been described as foot process spreading and retraction or as foot process effacement. This process by which podocytes change their cytoskeletal architecture appears to be a component of a common response of the podocyte to cellular injury, correlating with loss of normal filtration barrier selectivity and predicting the development of proteinuria in human disease and in experimental models (1, 2).

Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation.

No abstract

The Crk adaptor protein, which is encoded by two splice variants termed CrkI and CrkII, contains both SH2 and SH3 domains but no catalytic region. It is thought to function in signal transduction processes involved in growth regulation, cell transformation, cell migration, and cell adhesion. Although the function of Crk has been studied in considerable detail in cell culture, its biological role in vivo is still unclear, and no Crkknockout mouse model has been available. Therefore, we generated a complete null allele of Crk in mice by using the Cre-loxP recombination approach. The majority of Crk-null mice die at late stages of embryonic development, and the remainder succumb shortly after birth. Embryos lacking both CrkI and CrkII exhibited edema, hemorrhage, and cardiac defects. Immunohistochemical examination suggested that defects in vascular smooth muscle caused dilation and rupturing of blood vessels. Problems in nasal development and cleft palate were also observed. These data indicate that Crk is involved in cardiac and craniofacial development and that it plays an essential role in maintaining vascular integrity during embryonic development.