Characterization of the cytosolic tuberin-hamartin complex. Tuberin is a cytosolic chaperone for hamartin

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction and dermatological abnormalities. Mutations to either the TSC1 or TSC2 gene are responsible for the disease. The TSC1 gene encodes hamartin, a 130-kDa protein without significant homology to other known mammalian proteins. Analysis of the amino acid sequence of tuberin, the 200-kDa product of the TSC2 gene, identified a region with limited homology to GTPase-activating proteins. Previously, we demonstrated direct binding between tuberin and hamartin. Here we investigate this interaction in more detail. We show that the complex is predominantly cytosolic and may contain additional, as yet uncharacterized components alongside tuberin and hamartin. Furthermore, because oligomerization of the hamartin carboxyl-terminal coiled coil domain was inhibited by the presence of tuberin, we propose that tuberin acts as a chaperone, preventing hamartin self-aggregation

New rat model that phenotypically resembles autosomal recessive polycystic kidney disease

Numerous murine models of polycystic kidney disease (PKD) have been described. While mouse models are particularly well suited for investigating the molecular pathogenesis of PKD, rats are well established as an experimental model of renal physiologic processes. Han:SPRD-CY: rats have been proposed as a model for human autosomal dominant PKD. A new spontaneous rat mutation, designated wpk, has now been identified. In the mutants, the renal cystic phenotype resembles human autosomal recessive PKD (ARPKD). This study was designed to characterize the clinical and histopathologic features of wpk/wpk mutants and to map the wpk locus. Homozygous mutants developed nephromegaly, hypertension, proteinuria, impaired urine-concentrating capacity, and uremia, resulting in death at 4 wk of age. Early cysts were present in the nephrogenic zone at embryonic day 19. These were localized, by specific staining and electron microscopy, to differentiated proximal tubules, thick limbs, distal tubules, and collecting ducts. In later stages, the cysts were largely confined to collecting ducts. Although the renal histopathologic features are strikingly similar to those of human ARPKD, wpk/wpk mutants exhibited no evidence of biliary tract abnormalities. The wpk locus maps just proximal to the CY: locus on rat chromosome 5, and complementation studies demonstrated that these loci are not allelic. It is concluded that the clinical and renal histopathologic features of this new rat model strongly resemble those of human ARPKD. Although homology mapping indicates that rat wpk and human ARPKD involve distinct genes, this new rat mutation provides an excellent experimental model to study the molecular pathogenesis and renal pathophysiologic features of recessive PKD

Angiotensin II induced expression of transcription factors precedes increase in transforming growth factor-β1 mRNA in neonatal cardiac fibroblasts

Angiotensin II (ANG II), a potent vasoconstricting peptide, may act as a growth factor for cardiac muscle cells and induce hypertrophy. We examined the molecular phenotype of neonatal rat cardiac fibroblasts in relation to ANG II by studying the expression pattern of three transcription factors (Egr-1, c-fos and c-jun) and the transforming growth factor-β1 (TGF-β1). ANG II did not affect cell proliferation and growth of serum deprived neonatal cardiac fibroblasts as predicted from their DNA and protein contents. The expression of Egr-1 and c-fos was induced as early as 15 min that reached maximal levels at 45 min and declined thereafter, whereas c-jun was induced at 45 min and remained elevated up to 2 hrs of ANG II addition. ANG II up-regulated the expression of TGF-β1, which became apparent after 1 hr of incubation and reached a plateau between 16-48 hrs. Our results indicate that ANG II transiently stimulates the expression of transcription factors, which may up-regulate TGF-β1, that in turn could contribute to the process of myocardial extra-cellular matrix remodeling in hypertrophy

TSC2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin-hamartin complex

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction and dermatological abnormalities. Inactivating mutations to either of the TSC1 and TSC2 tumour suppressor genes are responsible for the disease. TSC1 and TSC2 encode two large novel proteins called hamartin and tuberin, respectively. Hamartin and tuberin interact directly with each other and it has been reported that tuberin may act as a chaperone, preventing hamartin self-aggregation and maintaining the tuberin-hamartin complex in a soluble form. In this study, the ability of tuberin to act as a chaperone for hamartin was used to investigate the tuberin-hamartin interaction in more detail. A domain within tuberin necessary for the chaperone function was identified, and the effects of TSC2 missense mutations on the tuberin-hamartin interaction were investigated to allow specific residues within the central domain of tuberin that are important for the interaction with hamartin to be pin-pointed. In addition, the results confirm that phosphorylation may play an important role in the formation of the tuberin-hamartin complex. Although mutations that prevent tuberin tyrosine phosphorylation also inhibit tuberin-hamartin binding and the chaperone function, our results indicate that only hamartin is phosphorylated in the tuberin-hamartin complex