DNA PK is another PIKK family member that plays a role in injury induced signaling and both ATM and DNA PK could phosphorylate histone H2AX on Serine139 following IR.
Phosphorylation of histone H2AX was examined in wild form and A T cells since DNA PK phosphorylates this page in the absence of ATM kinase activity, to investigate possible effects of CP466722 on DNA PK. While H2AX phosphorylation STAT inhibitors subsequent IR was inhibited by CP466722 or KU55933 in wild type cells, these ATM inhibitors did not prevent IR induced H2AX phosphorylation in A T cells, indicating a lack of detectable effects on DNA PK. In response to growth factor activation, AKT is activated by phosphorylation of threonine 308 by the PI3K pathway and serine 473 by other PIKK family members.
To show that CP466722 wasn’t inhibiting PI3K or PIKK household members, human fibroblasts were serum starved for 24h before being activated with IGF I either in the presence or absence of CP466722, KU55933 or Wortmannin. Serum starvation resulted in a nearly complete loss Gossypol 303-45-7 of AKT phosphorylation. These phosphorylation occasions were strongly induced upon addition of IGF I to serum starved cells and, not surprisingly, were strongly inhibited by the recognized PI3K inhibitor wortmannin.
No inhibition was observed with CP466722 or KU55933 treatment. Taken together, these results indicate that CP466722 prevents ATM kinase, but does not influence the cellular activity of PI3K or PIKK family members. Abl and Src Endosymbiotic theory kinases were identified in the original in vitro screens as possible targets of CP466722. We employed a mouse pre B cell model, to address cell cycle control whether CP466722 inhibits cellular Abl and Src kinases. In this system, the BCR Abl fusion protein is constitutively lively, driving autophosphorylation of residue tyrosine 245 and phosphorylation of a target CrkL on tyrosine 207. Src kinase undergoes intermolecular autophosphorylation of deposit tyrosine 416 on its activation loop to become fully activated.
In cells expressing BCR Abl, SRC kinases are activated and increased levels of Src phosphorylation have been reported suggesting that Src is active and undergoing autophosphorylation. As a control, CP466722 and KU55933 were shown to inhibit ATM kinase activity in the mouse pre B cells as demonstrated by disruption of p53 phosphorylation and p53 stabilization in reaction to IR. To determine whether the inhibitors affected Abl and Src kinase exercise, the mouse pre B cells were treated with CP466722, KU55933 or Imatinib as a positive control.
Needlessly to say, autophosphorylation of BCR Abl, endogenous Abl, and Abl dependent phosphorylation of CrkL were all found in get a grip on mouse pre B cells.