This result is consistent with our hypothesis that Xic2 phosphorylation in the

Because CDK2 activity is required for DNA replication initiation in the LSS and Xic2 is degraded during DNA polymerase switching following the CDK2 requirement, for these studies, it was necessary to use the HSS with single stranded DNA which supports DNA polymerase switching and Xic2 turnover, but does not require CDK activity, In the presence of either roscオーダー AP24534 ovitine or the purchase AT7519 CDK inhibitor p27, Xic2 turnover in the extract was significantly accelerated compared to the negative controls, Add itionally, in the presence of CDK inhibitors and in absence of DNA, the Xic2 protein bands remained unshifted indicating that the phosphoforms of Xic2 ob served in the absence of DNA and CDK inhibitors is due to CDK2 phosphorylation, To directly test the role of CDK2 phosphorylation on Xic2 stability, we mutated the three possible CDK con sensus sites within Xic2 individually and in combination to alanine to prevent phosphorylation, Mutation of residue Thr 58 had little effect on Xic2 turnover, while mutation of residues Ser 98 or Ser 131 both resulted in more efficient prote olysis of Xic2, Additionally, mutation of S98 abolished phosphoform 1 of Xic2 suggesting that phos phorylation of Xic2 at residue 98 is responsible for the shifted phosphoform 1 of Xic2 in the interphase extract, Simultaneous triple mutation of Xic2 at res idues T58, S98, and S131 resulted in a Xic2 mutant that did not exhibit a shift and was efficiently degraded in the interphase extract compared to wildtype Xic2, We further generated glutamic acid mutations of Xic2 at residues S98 and S131 to mimic constitutive phosphoryl ation and tested these Xic2 mutants for turnover.

Our studies indicated that the Xic2 S98E, S131E double mu tant and the S98E and S131E single point mutants were all degraded similarly or less efficiently than pan Akt 阻害剤 the wildtype Xic2 that is phosphorylated in the extract, This result is consistent with our hypothesis that Xic2 phosphorylation in the extract inhibits its turnover. Taken together, these studies suggest that CDK2 dependent phosphorylation of Xic2 at residues S98 and S131 negatively regulates its ubiquitination and degrad ation during interphase.

Xic2 is hyperphosphorylated at residues S78 S81 in a manner dependent upon single stranded DNA and a caffeine sensitive kinase In the interphase egg extract, in addition to the DNA independent phosphorylation of Xic2 by CDK2 cyclin, we have also observed an additional DNA dependent shift in the presence of single stranded DNA, The DNA dependent phosphorylation of Xic2 causes a doublet of Xic2 to appear in the presence of single stranded DNA with a slower migration than phosphoform 1 resulting from CDK2 phosphorylation of residue S98, Previous studies have indi cated that single stranded DNA in the interphase egg ex tract is replicated to form double stranded DNA ends which trigger activation of the checkpoint kinase, Ataxia Telangiectasia related protein, and induce the phosphorylation of Chk2, Consistent with these previous findings, we observed that the timing of the ssDNA dependent mobility shift of Xic2 coincided with the appearance of a Chk2 shift in the presence of double stranded DNA ends, Moreover, the Xic2 shifts observed in the presence of ssDNA or repli cated dsDNA ends was prevented by the addition of caffeine, a known inhibitor of Class IV Phosphatidylinositol 3 kinases such as ATR, suggesting that an ATM ATR like kinase may be responsible for phosphorylating Xic2 in the presence of ssDNA, We also noted a modest increase in Xic2 turnover in the presence of caffeine per haps indicating that the phosphorylation of Xic2 in the presence of ssDNA may be partially stabilizing, To further characterize the modification of Xic2 in the presence of ssDNA, we examined Xic2 in the presence of a variety of DNA templates in the Xenopus interphase egg extract.