Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus.

While the mechanisms that regulate actin dynamics in cellular motility are intensively studied, relatively little is known about signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at the cell membrane. The kidney podocyte provides a unique model for investigating these mechanisms since deletion of Nephrin or Neph1, two interacting components of the specialized podocyte intercellular junction, results in abnormal podocyte morphogenesis and junction formation. We provide evidence that extends the existing model by which the Nephrin-Neph1 complex transduces phosphorylation-mediated signals that assemble an actin polymerization complex at the podocyte intercellular junction. Upon engagement, Neph1 is phosphorylated on specific tyrosine residues by Fyn, which results in the recruitment of Grb2, an event that is necessary for Neph1-induced actin polymerization at the plasma membrane. Importantly, Neph1 and Nephrin directly interact and, by juxtaposing Grb2 and Nck1/2 at the membrane following complex activation, cooperate to augment the efficiency of actin polymerization. These data provide evidence for a mechanism reminiscent of that employed by vaccinia virus and other pathogens, by which a signaling complex transduces an outside-in signal that results in actin filament polymerization at the plasma membrane.Precisely regulated actin polymerization provides the motive force necessary for intercellular junction formation and contributes to defining the shape and polarity of the cell. Similarly, directed actin polymerization proximate to the leading edge of the plasma membrane drives cell motility and is required in the complicated dynamics of lamellipodia, filopodia, and other specialized membrane structures including invadopodia and podosomes (6). While substantial progress has been made in understanding the cellular and molecular mechanisms that determine actin dynamics in these model systems (32), less is known about membrane-based proximal signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at these sites.Kidney glomerular visceral epithelial cells or podocytes are necessary for maintaining the glomerular filtration barrier (reviewed in reference 20). When mature, these cells have a unique octopus-like structure comprised of a central cell body that gives off branching primary, secondary, and tertiary processes. The tertiary processes or "foot processes" attach the podocyte to the glomerular capillary basement membrane, where they surround the glomerular capillary and where they interdigitate to form a specialized intercellular junction called the "slit diaphragm." Here foot processes provide a necessary element of the permeability-selective glomerular filter, allowing passage of water, solutes, and other small macromolecules from the capillary lumen to the urinary space while restricting the flux of cells and larger macromolecules.The podocyte provides a unique model for investiga...

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).

Accumulating evidence suggests that mitogen-activated protein kinase signaling pathways form modular signaling complexes. Because the mixed lineage kinase dual leucine zipper-bearing kinase (DLK) is a large modular protein, structure-function analysis was undertaken to examine the role of DLK domains in macromolecular complex formation. DLK mutants were used to demonstrate that a DLK leucine zipper-leucine zipper interaction is necessary for DLK dimerization and to show that DLK dimerization mediated by the leucine zipper domain is prerequisite for DLK activity and subsequent activation of stress-activated protein kinase (SAPK). Heterologous mixed lineage kinase family members can be co-immunoprecipitated. However, the DLK leucine zipper domain interacted specifically only with the DLK leucine zipper domain; in contrast, DLK NH 2 -terminal region was sufficient to co-immunoprecipitate leucine zipper kinase and DLK. DLK has been shown to associate with the putative scaffold protein JIP1. This association occurred through the DLK NH 2 -terminal region and occurred independently of DLK catalytic activity. Although the DLK NH 2 -terminal region associated directly with JIP-1, this region did not interact directly with either DLK or leucine zipper kinase. Therefore, DLK may interact with heterologous mixed lineage kinase proteins via intermediary proteins. The NH 2 -terminal region of overexpressed DLK was required for activation of SAPK. These results provide evidence that protein complex formation is required for signal transduction from DLK to SAPK.

Mitogen-activated protein kinases (MAPKs)1 link a variety of extracellular signals to a diverse range of cellular responses such as proliferation, differentiation, and apoptosis. Three groups of mammalian MAPKs and the upstream kinases and stimuli that activate them have been studied most extensively. These include the p42/p44 MAPK (extracellular signal-regulated kinases, ERK1 and -2), that are generally activated by mitogens and differentiation-inducing stimuli, the stress-activated protein kinases (p46/p54 SAPK or JNK1, 2, or 3 and their splice isoforms) and the p38 mapk (1-6). The stress-activated protein kinases have also been termed JNK protein kinases because they were identified as the principal c-Jun N-terminal kinases. The JNK family kinases are activated by cell stress-inducing stimuli such as heat shock, UV irradiation, hyperosmolarity, and ischemia/reperfusion injury, and by activation of specific cell surface receptors (7-9). Studies performed in cell culture systems and investigation of genetic mutants in a number of model organisms has begun to establish a variety of distinct physiological roles for individual stress-activated protein kinases and their associated pathways. Indeed, distinct JNK family kinases have been implicated in multiple specific biological processes that include but are not limited to embryonic morphogenesis, regulation of the apoptotic response to cellular injury, regulation of thymocyte development and activation, and regulation of proliferation (10 -17).Experiments in yeast have provided evidence that MAP kinase pathways are assembled from a unique combination of protein kinases into distinct protein complexes or modules (18,19). The minimal MAPK module uniformly contains a MAP-KKK, a MAPKK, and a MAPK. The components of these modules interact via direct protein-protein interactions and/or are tethered to scaffolding proteins (20 -25). Importantly, assembly of MAPK modules appears to allow segregation of MAPK signaling components into units that are responsive to independent stimuli, that obtain appropriate subcellular targeting, that are insulated from similar modules, and that can regulate functionally distinct substrates (26 -28). Identification of the JIP family of JNK scaffolding proteins first established that mammalian cells organize JNK pathways into modules in a fashion similar to yeast (29). Three JIP family genes and several splice isoforms have been identified (30 -32). JIP proteins can form homo-and hetero-oligomers and are phosphoproteins. Whereas JIP3 is structurally distant from JIP1 and JIP2, each has been demonstrated to associate directly with a mixed lineage kinase, with MKK7 and with JNK (29,31,33). It has been proposed that JIP proteins facilitate mixed lineage kinase-dependent signal transduction to JNK possibly by aggregating the three components of a JNK module (30).Although a considerable amount of work has been done to establish the role of JIP1 in regulating JNK signaling, less is known about the mechanisms that govern this regulation. We previou...

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.

Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH 2 -terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.A large body of work has focused on signal transduction via protein kinases generically termed mitogen-activated protein kinases (MAPK) 1 that link a variety of extracellular signals to cellular responses as diverse as proliferation, differentiation, and apoptosis (reviewed in Refs. 1-3). Biochemical and genetic evidence has demonstrated that activation of a prototypical MAPK occurs through sequential activation of a series of upstream kinases: a serine/threonine MAPK kinase kinase (MAP-KKK) phosphorylates a dual specificity protein kinase (MAPKK or MKK or MEK) that in turn phosphorylates and activates a MAPK. Three groups of mammalian MAPKs and the upstream kinases and stimuli that activate them have been studied most extensively. These include the p42/p44 MAPK s (extracellular signal-regulated kinases, ERK1 and ERK2), that are generally activated by mitogens and differentiation inducing stimuli, the p46/p54 SAPK s (stress-activated protein kinases, SAPKs), and the p38 MAPK s. Stress-activated protein kinases were discovered as the principal c-Jun NH 2 -terminal kinases and therefore have also been termed JNKs. Distinct from ERK1 and ERK2, the SAPKs are predominantly activated by cell stress-inducing signals such as heat shock, ultraviolet irradiation, proinflammatory cytokines, hyperosmolarity, ischemia/reperfusion, and axonal injury.Like previously identified MAPK pathways in mammalian cells and yeast, the SAPK pathways were initially thought to lead in a lin...

It has been proposed that JNK-interacting proteins (JIP) facilitate mixed lineage kinase-dependent signal transduction to JNK by aggregating the three components of a JNK module. A new model for the assembly and regulation of these modules is proposed based on several observations. First, arti®cially induced dimerization of dual leucine zipper-bearing kinase (DLK) con®rmed that DLK dimerization is suf®cient to induce DLK activation. Secondly, under basal conditions, DLK associated with JIP is held in a monomeric, unphosphorylated and catalytically inactive state. Thirdly, JNK recruitment to JIP coincided with signi®cantly decreased af®nity of JIP and DLK. JNK promoted the dimerization, phosphorylation and activation of JIP-associated DLK. Similarly, treatment of cells with okadaic acid inhibited DLK association with JIP and resulted in DLK dimerization in the presence of JIP. In summary, JIP maintains DLK in a monomeric, unphosphorylated, inactive state. Upon stimulation, JNK±JIP binding af®nity increases while JIP±DLK interaction af®nity is attenuated. Dissociation of DLK from JIP results in subsequent DLK dimerization, autophosphorylation and module activation. Evidence is provided that this model holds for other MLK-dependent JNK modules.