One of the best defined functions our site of CK1�� is the Wnt induced phosphorylation of Dvl, a crucial component of the Wnt signaling cascades. In the first step, we analyzed the capacity of individual CK1�� mutants to phosphorylate Dvl in mammalian HEK293 cells. As shown in Figure 1b, WT CK1�� promoted the Inhibitors,Modulators,Libraries formation of phosphorylated and shifted Dvl2, whereas mutant forms did not. A partial shift was observed with the P6 mutant, but the P3 P4 mutants were indistinguishable when compared with the control transfected sample. Deletion of the C terminus of CK1e, which inhibits CK1e kinase activity when phosphorylated, did not affect these results. Importantly, using immunopre cipitation we were able to detect the presence of overex pressed Dvl2 or Dvl3, and both WT and mutated CK1e kinases in one protein complex, suggesting that the CK1e mutants still possess the ability to bind Dvl.

Phosphorylation of Dvl by CK1�� leads not only to the formation of PS Dvl but also to changes in the intracellu lar distribution of Dvl. Dvl is usually present in dynamic multiprotein aggregates called Dvl dots. Based on the cellular context and activity of CK1e, which promotes the dissolution of Dvl aggregates, Dvl Inhibitors,Modulators,Libraries is usually present either in dots or in an even distribution. WT Inhibitors,Modulators,Libraries CK1�� strongly promoted an even localization of Dvl2 Myc. In con trast, all of the analyzed mutants pro moted the formation maintenance of Dvl dots in COS7 cells and co localized with Dvl in these multiprotein complexes, similar to earlier observations with dominant negative CK1�� or CK1 inhibitors.

All CK1�� proteins were evenly distributed in the absence of Dvl. These data together show that despite the fact Inhibitors,Modulators,Libraries that CK1�� mutants bind and co localize with Dvl, they cannot phosphorylate Dvl or efficiently promote its even localization. contains two mutations, L39Q and S101R. These mutated sites are distant from each other not only in sequence but also spatially. While Leu 39 is located in the B strand rich N terminal lobe, Ser 101 is located at the C terminal end of helix I in the primarily helical C terminal subdomain. The S101R mutation is predicted to destabi lize and alter local protein structure, thus affecting the structural integrity of the four helix bundle, which forms the structurally conserved core of the C terminal subdo main. The last analyzed mutant, P4, contains three point mutations, L39Q, L49Q, and N78T.

Although the individ ually mutated sites are distant in primary Inhibitors,Modulators,Libraries sequence, when mapped onto the three dimensional structure of CK1�� they cluster into a very small area with a radius 10 that is located between strands four and five as well as into helix selleck chem Paclitaxel B of the N terminal lobe. Interestingly, all of these mutations surround a conserved predicted autophosphorylation site at Thr 44 in the N terminal cat alytic domain.