Adult male C57BL mice have been pre taken care of for one week with day-to-day ten mg/kg STI 571 or vehicle alone by means of intraperitoneal injection. On day seven animals acquired 4 injections i. p. in the VEGFR inhibition neurotoxin, 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine in saline or saline alone at 2 h intervals. Daily STI 571 injections continued up to 1 much more week following the last injection of MPTP. Animals had been processed and ready for biochemical and neurochemical assessment as previously described. GST pull down of K562 cell lysates with GST tagged complete length or truncated varieties of parkin exposed that N terminal domain of parkin interacts with c Abl. Pull down with GST tagged proteins of total length c Abl, and SH3, SH2, SH2 TK, TK DNA binding, DBD, and F actin domains of c Abl and lysates expressing FLAG parkin showed a strong interaction of parkin with complete length c Abl, and modest interaction with its truncated SH3 and SH2 domains.
Parkin Abl interaction is specific, considering the fact that FLAG parkin failed to interact with c Abl connected gene tyrosine kinase. In vitro c Abl kinase HDAC1 inhibitor assay with myc tagged constructs of parkin indicated that c Abl tyrosine phosphorylates only complete length parkin along with a construct lacking the ubiquitin like domain, suggesting that Y143 is substrate for c Abl. In vitro kinase reactions of GST fusion proteins of wild variety parkin, Y143F mutant parkin, ParN and ParC that has a 32 kDa lively tyrosine kinase domain of c Abl uncovered elevated tyrosine phosphorylation of wild sort parkin and ParN, but not of Y143F mutant parkin or ParC.
STI 571, a selective c Abl inhibitor, substantially decreased c Abl mediated tyrosine phosphorylation of GST parkin. In addition, parkin phosphorylation was not observed during the absence of c Abl. These results indicate that parkin specifically interacts with c Abl and that parkin is phosphorylated by c Abl at its N terminal Eumycetoma domain on Y143. In vitro ubiquitination assays working with recombinant GST parkin and SH2 TK c Abl revealed that c Abl mediated parkin phosphorylation substantially inhibited its E3 ubiquitin ligase action, as demonstrated by decreased parkin automobile ubiquitination. The phosphorylation resistant Y143F mutant of parkin showed small result on auto ubiquitination. Parkin mediated ubiquitination of AIMP2 was diminished while in the presence of c Abl, an effect that was blocked by STI 571. Parallel final results have been obtained utilizing an substitute parkin substrate FBP 1.
As a result, parkin mediated E3 ubiquitin ligase action is inhibited by c Abl mediated phosphorylation of parkin on Y143. Cellular tension induced by one hundred uM MPP, 250 uM H2O2, or a hundred uM DA activated PF299804 molecular weight c Abl in SH SY5Y cells, as measured by phospho c Abl amounts. Significant parkin phosphorylation and AIMP2 accumulation was also observed. STI 571 prevented parkin phosphorylation and AIMP2 accumulation.