-Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 g/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PANtreated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype. nucleocytoplasmic translocation; glomerulosclerosis; LIM domain; slit diaphragm; cell-cell contacts THE GLOMERULAR FILTRATION BARRIER is composed of a highly fenestrated endothelium, the glomerular basement membrane, and the podocyte. After a tightly orchestrated differentiation program, podocytes develop foot processes and assemble a specialized adherens junction, the slit diaphragm, which mediates contact between adjacent cells (44). In proteinuric diseases, regardless of the etiology, podocytes undergo marked morphological change. The actin cytoskeleton rearranges into a mat below the plasma membrane opposed to the glomerular basement membrane, slit diaphragm structures are lost, and the podocyte assumes a cuboidal shape. At a molecular level in both human biopsies and experimental models, this stereotypical morphological response is associated with changes in cytoplasmic, plasma membrane, and nuclear podocyte differentiation marker expression (5, 6). In addition, podocytes in some glomerular diseases also revert to a less differentiated phenotype more characteristic of the developing, rather than the fully differentiated, glomerulus (5). Appropriate treatment can restor...

Myosin II plays critical roles in events such as cytokinesis, chemotactic migration, and morphological changes during multicellular development. The amoeba Dictyostelium discoideum provides a simple system for the study of this contractile protein. In this system, myosin II filament assembly is regulated by myosin heavy chain (MHC) phosphorylation in the tail region of the molecule. Earlier studies identified an alpha-kinase, MHC kinase A (MHCK A), which phosphorylates three mapped threonine residues in the myosin tail, driving myosin disassembly. Using molecular and genomic approaches, we have identified a series of related kinases in Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization that includes a highly novel catalytic domain coupled to a carboxyl-terminal WD repeat domain. We have engineered, expressed, and purified a FLAG-tagged version of the novel kinase. In the present study, we report detailed biochemical and cellular studies documenting that MHCK B plays a physiological role in the control of Dictyostelium myosin II assembly and disassembly during the vegetative life of Dictyostelium amoebae. The presented data supports a model of multiple related MHCKs in this system, with different regulatory mechanisms and pathways controlling each enzyme.

Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least nine isoforms that are thought to participate in down-regulation of DG-based signalling pathways and perhaps activation of PA-stimulated signalling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs and a C-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated myosin heavy chain-protein kinase C (MHC-PKC), but our analysis clearly demonstrates that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust DGK activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system.