Proteolytic Cleavage and Nuclear Translocation of Fibrocystin Is Regulated by Intracellular Ca2+ and Activation of Protein Kinase C

Abstract

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca 2؉ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca 2؉ release or activation of protein kinase C. These results identify a novel Ca 2؉ -dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus. Autosomal recessive polycystic kidney disease (ARPKD)2 is a hereditary cause of kidney failure in infants and children. ARPKD affects 1 in 20,000 individuals and is characterized by aberrant epithelial cell proliferation, which causes cystic dilation of the renal collecting ducts, and abnormal development of intrahepatic bile ducts (1). Affected individuals present with bilateral kidney enlargement, intrauterine kidney failure, and oligohydramnios; the latter causes pulmonary hypoplasia and limb and facial abnormalities. Children who survive the perinatal period or develop ARPKD later in life develop chronic kidney disease and portal hypertension due to congenital hepatic fibrosis.ARPKD is caused by mutations of the polycystic kidney and hepatic disease gene 1 (PKHD1) located on chromosome 6 (2). The protein encoded by PKHD1 is termed fibrocystin (also called polyductin, or tigmin (3-5)). Fibrocystin is an ϳ500,000 dalton type I membrane protein comprised of a large N-terminal ectodomain, a single transmembrane segment, and a short C-terminal cytoplasmic domain. The ectodomain contains arrays of IPT (Ig-like, plexins, transcription factors) domains and PbH1 (parallel ␤-helix repeats) domains. The structure of fibrocystin suggests that it may function as a receptor for an as yet unidentified ligand. However, the authentic function of fibrocystin and the mechanism of signal transduction remain unknown. Recent studies suggest that the molecular pathogenesis of PKD may involve primary cilia and associated Ca 2ϩ -dependent signaling (6). Primary cilia are present on the apical membrane of renal tubular epithelial cells, and bending of the cilia in response to fluid flow shear stress elicits an increase in cytosolic Ca 2ϩ concentration ([Ca 2ϩ ]). Polycystin-1 and polycystin-2, which are affected in the autosomal dominant form of PKD, are located in primary cilia and are required for an initial Ca 2ϩ influx that is triggered by flu...

Epithelial-to-mesenchymal transition in cyst lining epithelial cells in an orthologous PCK rat model of autosomal-recessive polycystic kidney disease

“…Moreover, recent evidence suggests that fibrocystin/polyductin and polycystins are colocalized at the primary cilia (23,42,44). Fibrocystin/polyductin is reportedly related to the flow-stimulated intracellular calcium signaling pathway (12,22). These findings suggest that the pathophysiology of the PCK rat model may involve a common pathway that alters ciliary functions, thus leading to cyst formation.…”

Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

“…Although the gene responsible for ARPKD, PKHD1, has been identified [23][24][25] and its gene product, FPC, has been initially characterized, 27,28,30,31,39,40 the mechanisms by which PKHD1 causes disease phenotypes remain largely unknown. To study the disease mechanism and pathogenesis of ARPKD, we created a mouse that allows manipulation of Pkhd1, an animal model that recapitulates the human ARPKD phenotype.…”

Ciliary dysfunction in polycystic kidney disease: an emerging model with polarizing potential

“…Fibrocystin is also found at other subcellular localizations, such as cytosol, basal lateral membrane and nucleus (78)(79)(80)(81)(82)(83)110). Recent studies show that fibrocystin indirectly interacts with polycystin-2 at the cilium (47,48) (Figure 1).…”

“…The Pkhd1 transcript has a complex pattern of splice variants (76,77), and fibrocystin itself undergoes a complicated pattern of proteolytic processing (78,79). Fibrocystin has been shown to express in a tissue-specific manner, which varies with the stage of tissue development and cellular differentiation (80,81).…”