Biomedical subjects

Thomas Benzing

Publications and source records attributed to Thomas Benzing.

23 records · Page 2Linked to original sources

PubMed2003

Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination.

Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, leads to chronic renal failure in children. The genes mutated in NPHP1 and NPHP4 have been identified, and a gene locus associated with infantile nephronophthisis (NPHP2) was mapped. The kidney phenotype of NPHP2 combines clinical features of NPHP and polycystic kidney disease (PKD). Here, we identify inversin (INVS) as the gene mutated in NPHP2 with and without situs inversus. We show molecular interaction of inversin with nephrocystin, the product of the gene mutated in NPHP1 and interaction of nephrocystin with beta-tubulin, a main component of primary cilia. We show that nephrocystin, inversin and beta-tubulin colocalize to primary cilia of renal tubular cells. Furthermore, we produce a PKD-like renal cystic phenotype and randomization of heart looping by knockdown of invs expression in zebrafish. The interaction and colocalization in cilia of inversin, nephrocystin and beta-tubulin connect pathogenetic aspects of NPHP to PKD, to primary cilia function and to left-right axis determination.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Homodimerization and heterodimerization of the glomerular podocyte proteins nephrin and NEPH1.

Nephrin and NEPH1, the gene products of NPHS1 and NEPH1, are podocyte membrane proteins of the Ig superfamily. Similar to the nephrin knockout, mice lacking NEPH1 show severe proteinuria leading to perinatal death. To identify the ligand of NEPH1, the extracellular domain of NEPH1 was fused to human IgG. This NEPH1-Ig fusion protein labeled the glomerular capillary wall of mouse kidneys in a staining pattern identical to NEPH1 and nephrin, prompting speculation that that NEPH1 might form homodimers and/or heterodimers with nephrin. In coimmunoprecipitation and pull-down assays, the NEPH1-Ig fusion protein precipitated wild-type NEPH1 from overexpressing HEK 293T cells. Truncational analysis revealed that the adhesive properties were not confined to a single Ig domain of NEPH1. Fusion proteins containing two Ig domains of NEPH1 were sufficient to immobilize NEPH1, but they failed to interact with control protein containing the phylogenetically related PKD repeats of polycystin-1. NEPH1 also precipitated nephrin, a protein with eight Ig domains and a fibronectin-like domain. Truncational analysis of nephrin revealed a very similar mode of interaction, i.e., two nephrin Ig domains fused to human IgG precipitated either nephrin or NEPH1, but not the control protein. Both NEPH1 and nephrin interactions were strictly dependent upon posttranslational glycosylation, and bacterially expressed protein failed to bind NEPH1. These findings demonstrate that the Ig domains of NEPH1 and nephrin form promiscuous homodimeric and heterodimeric interactions that may facilitate cis- and trans- homodimerizations and heterodimerizations of these molecules at the glomerular slit diaphragm.

Animals↗

PubMed2003

Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling.

Mutations of NPHS1 or NPHS2, the genes encoding nephrin and podocin, as well as the targeted disruption of CD2-associated protein (CD2AP), lead to heavy proteinuria, suggesting that all three proteins are essential for the integrity of glomerular podocytes, the visceral glomerular epithelial cells of the kidney. It has been speculated that these proteins participate in common signaling pathways; however, it has remained unclear which signaling proteins are actually recruited by the slit diaphragm protein complex in vivo. We demonstrate that both nephrin and CD2AP interact with the p85 regulatory subunit of phosphoinositide 3-OH kinase (PI3K) in vivo, recruit PI3K to the plasma membrane, and, together with podocin, stimulate PI3K-dependent AKT signaling in podocytes. Using two-dimensional gel analysis in combination with a phosphoserine-specific antiserum, we demonstrate that the nephrin-induced AKT mediates phosphorylation of several target proteins in podocytes. One such target is Bad; its phosphorylation and inactivation by 14-3-3 protects podocytes against detachment-induced cell death, suggesting that the nephrin-CD2AP-mediated AKT activity can regulate complex biological programs. Our findings reveal a novel role for the slit diaphragm proteins nephrin, CD2AP, and podocin and demonstrate that these three proteins, in addition to their structural functions, initiate PI3K/AKT-dependent signal transduction in glomerular podocytes.

14-3-3 Proteins↗

PubMed2002

Interaction of 14-3-3 protein with regulator of G protein signaling 7 is dynamically regulated by tumor necrosis factor-alpha.

Regulators of G protein signaling (RGS) constitute a family of proteins with a conserved RGS domain of approximately 120 amino acids that accelerate the intrinsic GTP hydrolysis of activated Galpha(i) and Galpha(q) subunits. The phosphorylation-dependent interaction of 14-3-3 proteins with a subset of RGS proteins inhibits their GTPase-accelerating activity in vitro. The inhibitory interaction between 14-3-3 and RGS7 requires phosphorylation of serine 434 of RGS7. We now show that phosphorylation of serine 434 is dynamically regulated by TNF-alpha. Cellular stimulation by TNF-alpha transiently decreased the phosphorylation of serine 434 of RGS7, abrogating the inhibitory interaction with 14-3-3. We examined the effect of 14-3-3 on RGS-mediated deactivation kinetics of G protein-coupled inwardly rectifying K(+) channels (GIRKs) in Xenopus oocytes. 14-3-3 inhibited the function of wild-type RGS7, but not that of either RSG7(P436R) or RGS4, two proteins that do not bind 14-3-3. Our findings are the first evidence that extracellular signals can modulate the activity of RGS proteins by regulating their interaction with 14-3-3.

14-3-3 Proteins↗

PubMed2002

The polycystin-1 C-terminal fragment triggers branching morphogenesis and migration of tubular kidney epithelial cells.

Mutations of either PKD1 or PKD2 cause autosomal dominant polycystic kidney disease, a syndrome characterized by extensive formation of renal cysts and progressive renal failure. Homozygous deletion of Pkd1 or Pkd2, the genes encoding polycystin-1 and polycystin-2, disrupt normal renal tubular differentiation in mice but do not affect the early steps of renal development. Here, we show that expression of the C-terminal 112 amino acids of human polycystin-1 triggers branching morphogenesis and migration of inner medullary collecting duct (IMCD) cells, and support in vitro tubule formation. The integrity of the polycystin-2-binding region is necessary but not sufficient to induce branching of IMCD cells. The C-terminal domain of polycystin-1 stimulated protein kinase C-alpha (PKC-alpha), but not the extracellular signal-regulated kinases ERK1 or ERK2. Accordingly, inhibition of PKC, but not ERK, prevented polycystin-1-mediated IMCD cell morphogenesis. In contrast, HGF-mediated morphogenesis required ERK activation but was not dependent on PKC. Our findings demonstrate that the C-terminal domain of polycystin-1, acting in a ligand-independent fashion, triggers unique signaling pathways for morphogenesis, and likely plays a central role in polycystin-1 function.

Animals↗