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The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

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.

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 NH2-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.

The full-length mouse Indian hedgehog (Ihh) cDNA was cloned from an embryonic 17.5-day kidney library and was used to study the post-translational processing of the peptide and temporal and spatial expression of the transcript. Sequence analysis predicted two putative translation initiation sites. Ihh translation was initiated at both initiation sites when expressed in an in vitro transcription/translation system. Expression of an Ihh mutant demonstrated that the internal translation initiation site was sufficient to produce the mature forms of Ihh. Ihh post-translational processing proceeded in a fashion similar to Sonic and Drosophila hedgehog; the unprocessed form underwent signal peptide cleavage as well as internal proteolytic processing to form a 19-kDa amino-terminal peptide and a 26-kDa carboxyl-terminal peptide. This processing required His313 present in a conserved serine protease motif. Ihh transcript was detected by in situ RNA hybridization as early as 10 days postcoitum (dpc) in developing gut, as early as 14.5 dpc in the cartilage primordium, and in the developing urogenital sinus. In semiquantitative reverse transcription-polymerase chain reaction experiments, Indian hedgehog transcript was first detected in the mouse metanephros at 14.5 dpc; transcript abundance increased with gestational age, becoming maximal in adulthood. In adult kidney, Ihh transcript was detected only in the proximal convoluted tubule and proximal straight tubule.

Because the catalytic domain of dual leucine zipper-bearing kinase (DLK) bears sequence similarity to members of the mitogen-activated protein (MAP) kinase kinase kinase subfamily, this protein kinase was investigated for its ability to activate MAP kinase pathways. When transiently transfected and overexpressed in either COS 7 cells or NIH3T3 cells, wild type DLK potently activated p46(SAPK) (SAPK/JNK) but had no detectable effect in activating p42/44(MAPK). DLK also activated p38(mapk) when overexpressed in NIH3T3 cells. A catalytically inactive point mutant of DLK had no effect in these experiments. Consistent with its specificity in activating SAPK, DLK activated Elk-1 but not Sap1a-mediated transcription. In NIH3T3 cells, activation of SAPK by v-Src was markedly attenuated by coexpression of K185A, a catalytically inactive mutant of DLK, suggesting that this mutant could function in a dominant negative fashion in a pathway that leads from v-Src to SAPKs. In a series of co-transfection experiments, activation of p46(SAPK) by DLK was not inhibited by dominant negative mutants of Rac1 and Cdc42Hs, PAK65-R, or PAK65-A, but was attenuated by MEKK1(K432M). DLK(K185A) did not inhibit the ability of constitutively active MEKK1 to activate SAPK. Moreover, K185A significantly inhibited the activation of SAPK by constitutively active V-12 Rac1 and V-12 Cdc42Hs. These results suggest that DLK lies distal to Rac1 and/or Cdc42Hs but proximal to MEKK1 in a pathway leading from v-Src to SAPKs activation.

The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protein kinases, was examined in the nervous system. DLK transcript expression in the nervous system was predominantly neuronal. DLK protein was present in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had differing characteristics. Cytosolic DLK existed in both a phosphorylated and dephosphorylated state; DLK associated with plasma membrane existed in the dephosphorylated state only. On nonreducing SDS-polyacrylamide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while membrane associated DLK migrated with an apparent Mr >/= 260,000. Similarly, DLK transiently expressed in COS 7 cells autophosphorylated in vivo and migrated at approximately 260 kDa when separated by nonreducing SDS-polyacrylamide gel electrophoresis. In cotransfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-Delta520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conclusion that DLK formed covalently associated homodimers in overexpressing COS 7 cells. In aggregating neuronal-glial cultures, depolarization of plasma membrane lead to dephosphorylation of DLK. Treatment of aggregates with 5 nM or 200 nM okadaic acid lead to a shift in electrophoretic mobility consistent with phosphorylation of DLK. Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulin-dependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.

Molecular cloning using a degenerate oligonucleotide-based polymerase chain reaction was undertaken to test the possibility that novel, developmentally regulated protein kinases are expressed in the embryonic mouse kidney. Several receptor tyrosine kinase and serine/threonine kinase cDNA clones were identified. One of these, designated DLK, represented a novel gene product whose 3.6-kilobase transcript was expressed in a tissue-specific and developmentally regulated fashion. Several clones encoding the entire open reading frame were isolated and sequenced. The identified open reading frame encodes an 888-amino acid polypeptide that defines a new subfamily within the mixed lineage protein kinase family. Sequence analysis revealed: 1) a kinase catalytic domain most characteristic of serine/threonine kinases but hybrid between members of the family of microtubule-associated protein kinase kinase kinases and the fibroblast growth factor receptor family; 2) two putative alpha-helical leucine zipper motifs separated by a 25-amino acid charged intermediate segment but lacking an NH2-terminal basic domain; and 3) COOH-terminal and NH2-terminal proline-rich domains suggestive of src homology 3 (SH3) domain binding regions. Rabbit polyclonal immune sera generated against a carboxyl-terminal bacterial fusion protein recognized a protein with an apparent molecular mass of 130 kDa in COS 7 cells that were transiently transfected with a full-length DLK cDNA expression vector. Moreover, COS 7 cells transiently transfected with an epitope-tagged DLK expression vector expressed protein with an apparent molecular mass of 130 kDa that became autophosphorylated on serine and threonine in an in vitro kinase assay.

Podocytes are specialized epithelial cells with delicate interdigitating foot processes which cover the exterior basement membrane surface of the glomerular capillary. They are in part responsible for the extraordinary charge and size filtration characteristics of the glomerulus. To better understand disease processes affecting the glomerular filter, we searched for proteins with relative specificity to the podocyte using a monoclonal antibody strategy. The first such protein characterized (designated glomerular epithelial protein 1 (GLEPP1)) is a membrane protein-tyrosine phosphatase (PTPase) with a large extracellular domain containing eight fibronectin type III-like repeats, a hydrophobic transmembrane segment, and a single PTPase domain. The GLEPP1 PTPase domain shows homology with two other single domain transmembrane PTPases (PTP beta and Drosophila central nervous system PTP10D). This homology includes 2 cysteines in the PTPase domain not present in intracellular or tandem domain membrane PTPases. GLEPP1 PTPase protein is distributed to the podocyte foot processes themselves. RNase protection assay shows that GLEPP1 mRNA is also present in brain. By analogy with the CD45 PTPase of T cells, we expect that this receptor might play a role in maintaining foot process structure and/or function by regulating tyrosine phosphorylation of podocyte proteins.

Cytokines such as tumor necrosis factor alpha (TNF) profoundly affect endothelial cell function, promoting for example interaction with leukocytes and inducing a procoagulant phenotype. Changes of this nature are likely to be central to the proinflammatory effects of TNF. In order to elucidate molecular mechanisms by which TNF alters endothelial cell function we utilized differential plaque hybridization to identify TNF-responsive genes. Forty TNF-inducible cDNAs were identified which on cross-hybridization were found to arise from six unique genes. DNA sequencing of these cDNAs revealed two encoded known cytokine-induced genes, endothelial leukocyte adhesion molecule 1 and neutrophil chemotactic factor. One of the cDNAs encodes a recently described monocyte-specific chemotactic factor not previously associated with endothelium. The production of a monocyte chemotaxin by cytokine-activated endothelium has important implications for understanding the role of the vessel wall in disease states such as atherosclerosis and may also in part explain the indirect angiogenic activity of TNF. The three other cDNAs are completely novel as judged by data bank searches of partial DNA sequences and remain unidentified. On exposure of endothelial cells to TNF there is a rapid and substantial increase in levels of mRNA encoding the six genes, which are further superinduced by cycloheximide. Thus these represent primary response genes as their induction does not depend on protein synthesis. Interleukin-1 beta and lipopolysaccharide are also potent inducers. Nuclear run-on studies revealed that in most cases induction by TNF is mediated largely at the transcriptional level.