Genotoxin-induced degradation of Akt is independent of the proteasome and is abrogated by maintenance of tyrosine phosphorylation

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Cell survival following genotoxic stress results in the acquisition of a selective growth advantage that predisposes these cells to transformation. Dysregulation of survival pathways such as that involving Akt may contribute to the development of the transformed phenotype. Certain forms of hexavalent chromium (Cr(VI)) are respiratory carcinogens and genotoxins. It has been reported that certain DNA damaging agents initially activate Akt but this activation is followed by down-regulation of both phosphorylation state and total protein expression. Our overarching objective is to determine the role of Akt in cell survival after genotoxin exposure. In human lung fibroblasts (HLF), 24 h exposure to 1-6 μM Cr(VI) was shown to result in a dose-dependent decrease in both phospho-ser473 Akt and total Akt expression, as well as Akt activity as measured by phosphorylation of the substrate GSK3β in vitro. The aim of this study was to determine the mechanisms underlying the Cr(VI)-dependent decrease in Akt expression in HLF cells. Involvement of the proteasome was ruled out since inhibition by MG132 (10-25 μM) for 24 hours did not reverse Cr(VI)-induced degradation of Akt in HLF cells. We have shown that maintenance of tyrosine phosphorylation through protein tyrosine phosphatase (PTP) inhibition activates survival pathways such as that involving Akt. Sodium orthovanadate (SOV, 10μM), a broad range protein tyrosine phosphatase inhibitor, when co-incubated with Cr(VI) (1-6μM) resulted in the abrogation of the Cr(VI)-induced decrease in both total Akt and phospho-ser473 Akt expression as well as Akt activity, suggesting a role for protein tyrosine phosphatases in Akt downregulation. Furthermore, we have shown that 10μM SOV can override Cr(VI)-induced G2/M arrest, as well as increase Cr(VI)-induced clonogenic survival in HLF cells, thereby suggesting a role for tyrosine phosphorylation in overriding genotoxin death signals. These results suggest that the Cr(VI)-induced downregulation of Akt may involve tyrosine phosphatase activity, as suggested by the fact that the Cr(VI)-induced decrease in Akt expression and activation can be reverted by SOV. The Ser/Thr protein phosphatases, PP2A and PP1, have been implicated in the dephosphorylation of Akt induced by H₂O₂. Therefore downregulation of Akt could be due to Ser/Thr phosphatase inactivation by tyrosine phosphatase-dependent mechanisms. Akt appears to be at the nexus between death and survival after genotoxic exposure. Understanding the mechanisms involved in Akt down-regulation in response to genotoxic stress would provide a better understanding of why some cells survive genotoxic insult and eventually progress to a malignant phenotype. Supported by NIH grants ES05304 and ES09961 to SRP.