PMC:3216504 / 17852-19678
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3216504","sourcedb":"PMC","sourceid":"3216504","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3216504","text":"We characterized the function of these mutations by generating the equivalent mutations in mouse Zic2 proteins, and assessing their activities in vitro. The size of the mutant Zic2 proteins was mostly comparable to that of the wild type protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Figure 6B). However, the band corresponding to Zic2-S444R also contained a fast migrating component in preparations from both transfected mammalian cells and from in vitro translation (Figure 6B and data not shown). The nuclear localization capacity of the mutant proteins was significantly reduced for R409P (P \u003c 0.001 in chi-square test), but not for A95T or S444R (Figure S2). Zic2 can be a transcriptional activator3132. We assessed the transactivating activity of the mutant proteins by co-transfecting mammalian cells with expression vectors that express the proteins. The Zic2-R409P protein, but not the Zic2-A95T or Zic2-S444R proteins had significantly reduced transactivating capacity compared to the wild-type protein (P \u003c 0.001, Figure 6C); the normalized activation capacity of Zic2-R409P was approximately 20% that of the wild-type protein. When we examined the effect of the R409P mutation on the capacity to bind a high-affinity Zic-binding sequence, Zic2-R409P showed lower binding affinity to the target sequences than wild-type Zic2 (Figure 6D and 6E). Zic2 is known to complex with the DNA-PK catalytic subunit and RHA33. As shown in immunoprecipitation experiments (Figure 6F), the binding affinity of Zic2-R409P to DNA-PK, but not to RHA, was reduced compared to wild-type Zic2. Taken together, these results suggested that the R409P mutation dampens the transcriptional activation capacity of Zic2 by altering the properties of the Zic2-containing molecular complex.","tracks":[{"project":"2_test","denotations":[{"id":"22355535-11053430-138031319","span":{"begin":756,"end":758},"obj":"11053430"},{"id":"22355535-15207726-138031320","span":{"begin":756,"end":760},"obj":"15207726"},{"id":"22355535-17251188-138031321","span":{"begin":1475,"end":1477},"obj":"17251188"}],"attributes":[{"subj":"22355535-11053430-138031319","pred":"source","obj":"2_test"},{"subj":"22355535-15207726-138031320","pred":"source","obj":"2_test"},{"subj":"22355535-17251188-138031321","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#c2ec93","default":true}]}]}}