ATM f showed multiple putative phosphorylation sites cdk5. We have purified an in vitro Luteolin Luteolol kinase Cdk5 by incubation of purified ATM Cdk5 in complex with the p25 activator. Co-incubation with Cdk5 ATM leads to phosphorylation of ATM. We tested a series of GST fusion proteins, ATM and showed that the GST robust in vitro by Cdk5 phosphorylates ATM4. The mutation of serine 794 phosphorylation by Cdk5, the abolition alanine, suggesting that Cdk5 directly Recogn t S794 ATM in vitro. To demonstrate that ATM is a substrate for Cdk5 in cells, we raised an antique Body, the phospho-specific ATM with a phospho-synthetic peptide corresponding to residues of human ATM in S794. This antique Body, specifically recognized the phospho ATM wild-type ATM phosphorylated at S794 by Cdk5 where in vitro, but not S794A mutant.
We have then shown that the antique Body recognized phospho ATM very low background signal when the ATM was expressed alone in HEK293 cells. Coexpression of CDK5/p25 led to a strong signal phospho S794. Interestingly, the mutation of AB1010 serine 1981 to alanine does not affect the phosphorylation at S794, suggesting that Cdk5 phosphorylates directly ATM-independent Ngigen S1981 S794. Regulate when considering how the phosphorylation of ATM by Cdk5 can ATM activity T, we overexpressed wild-type ATM with or without co-expression of Cdk5/activator in HEK293 cells, immunpr Zipitiert ATM, ATM activity t and determined by assaying the kinase in vitro with a commercial substrate PHAS-I. Co-expression of Cdk5 strongly by weight are activated, w During ATM S794A mutant made in response to Cdk5.
Weight was compared with phospho-mimetic S794D mutant ATM kinase activity Tons more. In addition, the overexpression led by CDK5/p35 or Cdk5 / p25 in HEK293 cells resulted in a robust activation of endogenous ATM. Sun S794 phosphorylation activates ATM kinase activity t. We tested whether Cdk5 plays a role Upon activation of ATM DNA-Sch Tion damage original zerebell Re after granule neurons due to their selective degeneration of ATM deficiency. CGN for 7 days from postnatal day 6-7 rats were cultured, exposed to DNA-Sch The agents which are known to cause Bezirksschulr-run in postmitotic neurons19-21. Compared to an untreated control group, all three agents caused DNA-Sch Ending a strong activation of both ATM and Cdk5 kinase activity of t.
In contrast, classical stausporine agent for the induction of apoptosis, which do not usually to the CBD will not be activated, either Cdk5 or ATM. Tests of general inducers of cell death showed that several of them resembled erm Both Cdk5 and ATM. Interestingly, serum / potassium deprivation, on loan St other widely used model of CGN apoptosis not directly by DNA-Sch Endings, activates Cdk5 activity T without adversely caning of the ATM. The subcellular Re fractionation showed that S / K withdrawal of a galvanized Siege to activate Cdk5 only in the cytoplasm causes and not to activate Cdk5 in the nucleus. Together, these results indicate that activation of ATM Cdk5 and in the post-mitotic neurons, a response mode to represent a wider range of signals.
Since camptothecin is very effective in the activation of Cdk5 and ATM under our experimental conditions, we have used as a model for future studies. Because protease Calpa Damage22 activated by DNA and involved in the conversion of p35 to Cdk5 st Strongest activator p2523, we tested whether CPT increased p25 Cdk5 over Activated ht. CPT treatment caused a rapid and transient erh Increase of p25 levels correlated with increased Activity hter t Calpa Given as the split-fodrin α. CPT-induced Tian et al. Page 2 Nat Cell Biol author manuscript in PMC 12th October 2009. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript activation of Cdk5 and p25 in parallel rise preceded by the activation of the nuclear ATM. The inhibition of Calpa Not