Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.

Summary Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G2/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of post-transcriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2, phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the post-transcriptional regulation of gene expression as part of the DNA damage response in cancer cells.

Nephronophthisis is the most common genetic cause of end-stage renal failure during childhood and adolescence. Genetic studies have identified disease-causing mutations in at least 11 different genes (NPHP1–11), but the function of the corresponding nephrocystin proteins remains poorly understood. The two evolutionarily conserved proteins nephrocystin-1 (NPHP1) and nephrocystin-4 (NPHP4) interact and localize to cilia in kidney, retina, and brain characterizing nephronophthisis and associated pathologies as result of a ciliopathy. Here we show that NPHP4, but not truncating patient mutations, negatively regulates tyrosine phosphorylation of NPHP1. NPHP4 counteracts Pyk2-mediated phosphorylation of three defined tyrosine residues of NPHP1 thereby controlling binding of NPHP1 to the trans-Golgi sorting protein PACS-1. Knockdown of NPHP4 resulted in an accumulation of NPHP1 in trans-Golgi vesicles of ciliated retinal epithelial cells. These data strongly suggest that NPHP4 acts upstream of NPHP1 in a common pathway and support the concept of a role for nephrocystin proteins in intracellular vesicular transport.