Identification Of JAK1as A Novel Regulator For PCP

The dysregulation of biological process during embryonic development results inkinds of disease. One of the impornt biological process is the establishment of planarcell polarity (PCP), like the the orientation of bristles and the orientation of hair cellsin the inner ear. PCP pathway regulates cell polarity in nearly all of the tissues andorgans, the mutation of PCP regulators result in severe disease including Robinowsyndrome, Brachydactyly Type B1, and Neural tube defects.Neural tube defects (NTDs) are a group of malformations of the brain and spinalcord that occurs very early in embryonic development. NTDs include anencephalus,encephalocele, rachischisis and craniorachischisis according to the unclosed positionand the severity. Recent studies in NTD model organisms indicate that PCP pathwayespecially Vangl2play a key developmental role in neural tube closure, which hasbeen a new hot area of NTD research.PCP was first noted in Drosophila, which provided key mechanistic insights intoPCP signaling. The stereotyped arrangement of sensory hairs on the wing, abdomen,and thorax (notum) as well as the ommatidia in the eye all serve as models of planarpolarity. The core PCP proteins identified in Drosophila includeVanGogh(Stbm/Vang), Ror2, Frizzled(FZ), Dishevelled(Dsh/Dvl), Flamingo(Fmi),Prickle(Pk), and Diego(Dgo). The Genetic and biochemical analysis showd that thecore PCP proteins formed a complex compounds and the mutations or misexpressionof the core PCP genes resulted in the loss of polarity in these tissues. Recent researchhas shown that the intracellular distribution of Vangl2is asymmetry, Wntsdose-dependently induce Vangl2phosphorylation of serine/threonine residues andVangl2activities depend on its levels of phosphorylation. Wnt signaling gradientsestablish planar cell polarity by inducing Vangl2phosphorylation through Ror2.Despite the critical roles of Vangl2phosphorylation and asymmetricaldistribution in vertebrate morphogenesis and development, little is known about thebiology and cytology mechanism of Vangl2. The researches on upstream signalregulation and downstream signal transmission of Vangl2have great significance on revealing the mechanism of PCP pathway. Therefore, we started the research with theidentification of the new PCP components. The co-immunoprecipitation (co-IP) assaywas employed to isolate Vangl2protein complex and the immunoprecipitate wassubjected to MALDI TOF mass spectrometry (MS) for protein identification. Afteranalysis and investigation of those candidates, we focused on JAK1. JAK1is a kindof Non-receptor tyrosine kinase and is familiar with JAK-STAT pathway, whichinvolved in cell proliferation, differentiation, apoptosis and immunoregulation andmany other important biological processes. Recent studies have shown that theJAK/STAT pathway is also required in cell migration and convergent extension,which suggest that JAK1may play a role in PCP pathway. We first used co-IP andimmunofluorescence stain experiments to confirm that there is a direct interaction andcolocalization between JAK1and Vangl2in cell membrane. We then found thatJAK1can tyrosine-phosphorylated Vangl2and proved that the serine/threoninephosphorylation of Vangl2is negatively correlated with the tyrosine phosphorylationand the bingding level between Vangl2and JAK1. On the other hand, JAK1promotesthe interaction between Vangl2and Ror2. This contradiction results suggest that JAK1may regulate other components like Ror2and Wnt5a to further influence Vangl2.We found a breakthrough on Ror2. Our experiment results showed that JAK1can combine and tyrosine-phosphorylated Ror2, and Wnt5a time-course can regulatethe tyrosine phosphorylation of Ror2as well as the bingding between JAK1and Ror2:As the time-course went by, the binding capacity of JAK1was weakening, thetyrosine phosphorylation level of Ror2declined while the interaction between Ror2and Vangl2enhanced. These changes disappeared after treated with specific kinaseinhibitors of JAK1.Taken together, our finding indicated that JAK1act as a novel regulator of PCPpathway and provided further clues to elucidate the phosphorylation regulation ofVangl2and the signaling crosstalk between JAK1-STAT and PCP pathway. Ourresearch may bring new views in pathogenesis and treatment of PCP-related diseases.