Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway.

The Hedgehog (Hh) signaling pathway plays crucial roles in embryonic development and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Aberrant activation of Hh signaling is associated with various developmental abnormalities and several types of cancer. Genetic and biochemical studies ascertain serine/threonine kinase Fused (Fu) as a protein involved in Hh signaling in Drosophila. However, the role of Fu is not fully conserved in mammals suggesting involvement of other kinases in the mammalian Hh signaling pathway. In search of potential homologues to Drosophila and human Fu, we have cloned human serine/threonine kinase ULK3 and assessed its ability to regulate GLI transcription factors, mediators of SHH signaling. We demonstrate that ULK3 enhances endogenous and over-expressed GLI1 and GLI2 transcriptional activity in cultured cells, as assessed by GLI-luciferase reporter assay. Besides that, ULK3 alters subcellular localization of GLI1, as assessed by immunofluorescent staining and immunoblotting assays. We show that ULK3 is an autophosphorylated kinase and phosphorylates GLI proteins in vitro. We also demonstrate that ULK3 catalytical activity is crucial for its function in SHH pathway. We show that ULK3 is widely expressed and its expression is higher in a number of tissues where Shh signaling is known to be active. Our data suggest that serine/threonine kinase ULK3 is involved in the SHH pathway as a positive regulator of GLI proteins.

Identification of the gene transcription repressor domain of Gli3.

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

Characterization of the physical interaction of Gli proteins with SUFU proteins.

The Hedgehog signaling pathway is involved in both development and cancer induction in a wide range of organisms. The end point of the Hedgehog signal-transduction cascade is the Gli/Ci, zinc-finger transcription factors. Proteins such as Fused, Suppressor of fused (SUFU), Costal-2, and protein kinase A are essential for regulation of Gli/Ci processing, activity, and localization. Coimmunoprecipitation and Far Western assays, coupled with truncation analysis and mutagenesis have been used to define the region of interaction between Gli proteins and SUFU. We identify a novel motif SYGH in Gli/Ci family proteins, which is required for the interaction with SUFU. Mutational studies revealed that Gly(122) and His(123) are crucial for binding to SUFU, suggesting the importance of hydrophobicity for the correct binding conformation. Functional analysis revealed that the activity of GLI transcription factors with mutations in this motif is no longer suppressed by co-expression of SUFU. Moreover, we have found that a C-terminal 19-amino acid deletion in SUFU (delta465) is sufficient to abrogate interaction with GLI1. Interestingly, this SUFU mutant localizes in the nucleus, most probably because it is not efficiently sequestered in the cytoplasm. Taken together, we identified a novel motif in the Gli/Ci family of proteins that is essential both for protein-protein interaction with SUFU and for functional repression of GLI1 by SUFU.

Missense mutations in the extracellular domain of the human neural cell adhesion molecule L1 reduce neurite outgrowth of murine cerebellar neurons.

Mutations in L1CAM, the gene encoding the transmembrane multifunctional neuronal adhesion molecule L1, are associated with neurodevelopmental disorders including X-linked hydrocephalus and mental retardation. Some amino acid substitutions in various extracellular domains of L1 are known to affect posttranslational processing of the protein or its homophilic and heterophilic interactions. It is largely unknown, however, how these mutations result in neurodevelopmental disturbances and whether the effects of mutations on neurodevelopment can be modeled in vitro. We stably expressed full-length human wild type L1 and the known pathogenic missense mutations I179S, R184W, Y194C, and C264Y in NIH-3T3 cells. L1 protein synthesis, glycosylation pattern, and subcellular localization were analyzed. Neurite outgrowth of primary murine cerebellar neurons was measured after 23 hrs of co-cultivation using transfected NIH-3T3 cells as substrate. Like wild type L1, L1 protein with I179S or Y194C mutations was localized on the surface of the transfected substrate cells, but this was not the case with R184W or C264Y mutations. All four mutations were associated with reduced stimulation of neurite outgrowth. Measurement of neurite outgrowth on transfected substrate cells may be a suitable model for studying neurodevelopmental disturbances.