Cyclin-Dependent Kinase 5 (CDK5)-Mediated Phosphorylation of Upstream Stimulatory Factor 2 (USF2) Contributes to Carcinogenesis.

The transcription factor USF2 is supposed to have an important role in tumor development. However, the regulatory mechanisms contributing to the function of USF2 are largely unknown. Cyclin-dependent kinase 5 (CDK5) seems to be of importance since high levels of CDK5 were found in different cancers associated with high USF2 expression. Here, we identified USF2 as a phosphorylation target of CDK5. USF2 is phosphorylated by CDK5 at two serine residues, serine 155 and serine 222. Further, phosphorylation of USF2 at these residues was shown to stabilize the protein and to regulate cellular growth and migration. Altogether, these results delineate the importance of the CDK5-USF2 interplay in cancer cells.

Corrigendum: Protein kinases as switches for the function of upstream stimulatory factors: implications for tissue injury and cancer.

[This corrects the article on p. 3 in vol. 6, PMID: 25741280.].

Protein kinases as switches for the function of upstream stimulatory factors: implications for tissue injury and cancer.

The upstream stimulatory factors (USFs) are regulators of important cellular processes. Both USF1 and USF2 are supposed to have major roles in metabolism, tissue protection and tumor development. However, the knowledge about the mechanisms that control the function of USFs, in particular in tissue protection and cancer, is limited. Phosphorylation is a versatile tool to regulate protein functions. Thereby, phosphorylation can positively or negatively affect different aspects of transcription factor function including protein stability, protein-protein interaction, cellular localization, or DNA binding. The present review aims to summarize the current knowledge about the regulation of USFs by direct phosphorylation and the consequences for USF functions in tissue protection and cancer.

GSK3ß-dependent phosphorylation alters DNA binding, transactivity and half-life of the transcription factor USF2.

The upstream stimulatory factor 2 (USF2) is a regulator of important cellular processes and is supposed to have also a role during tumor development. However, the knowledge about the mechanisms that control the function of USF2 is limited. The data of the current study show that USF2 function is regulated by phosphorylation and identified GSK3ß as an USF2-phosphorylating kinase. The phosphorylation sites within USF2 could be mapped to serine 155 and threonine 230. In silico analyses of the 3-dimensional structure revealed that phosphorylation of USF2 by GSK3ß converts it to a more open conformation which may influence transactivity, DNA binding and target gene expression. Indeed, experiments with GSK-3ß-deficient cells revealed that USF2 transactivity, DNA binding and target gene expression were reduced upon lack of GSK3ß. Further, experiments with USF2 variants mimicking GSK3ß phosphorylated USF2 in GSK3ß-deficient cells showed that phosphorylation of USF2 by GSK3ß did not affect cell proliferation but increased cell migration. Together, this study reports a new mechanism by which USF2 may contribute to cancerogenesis.

The upstream stimulatory factor USF1 is regulated by protein kinase CK2 phosphorylation.

The upstream stimulatory factors 1 (USF1) and 2 (USF2) are transcription factors which bind to E-box motifs of various promoters regulating a variety of different cellular processes. Only little is known about the regulation of USFs. Here, we identified protein kinase CK2 as an enzyme that phosphorylates USF1 but not USF2. Using deletion mutants and point mutants we were able to identify threonine 100 as the major phosphorylation site for CK2. It is well known that USF1 and USF2 form hetero-dimers. Binding studies revealed that the inhibition of CK2 kinase activity by a specific inhibitor enhanced binding of USF1 to USF2. Furthermore, transactivation studies showed that the inhibition of CK2 phosphorylation of USF1 stimulated transcription from the glucokinase promoter as well as the fatty acid synthetase promoter but not from the heme oxygenase-1 promoter. Thus, we have shown for the first time that CK2 phosphorylation of USF1 modulates two functionally important properties of USF1, namely hetero-dimerization and transactivation.

Opposite expression of the antioxidant heme oxygenase-1 in primary cells and tumor cells: regulation by interaction of USF-2 and Fra-1.

Heme oxygenase-1 is the rate-limiting enzyme for the degradation of the prooxidant heme. Previously, we showed that an E-box within the HO-1 promoter is crucial for the regulation of HO-1 expression in primary hepatocytes. Further to investigate the importance of this E-box, we determined the regulatory capacity of the E-box-binding factor USF-2 in primary cells in comparison with transformed cell lines. We found that HO-1 expression was inhibited by USF-2 in primary cells, whereas it was induced in tumor cell lines. Mutation of either the E-box or the AP-1 site within the HO-1 promoter only partially affected the USF-dependent regulation. However, this regulation was dramatically reduced in tumor cells and completely abolished in primary cells transfected with an HO-1 promoter construct containing mutations in both the E-box and the AP-1 site, suggesting that AP-1 factors and USF-2 may act in a cooperative manner. Indeed, protein-protein interaction studies revealed that USF proteins interacted with Fra-1. Further, the USF-dependent HO-1 promoter activity was not detectable with an USF-2 mutant lacking residues of the USF-specific region (USR) or the transactivation domain encoded by exon 4. Together, these data suggest that USF-2 has opposite regulatory roles for HO-1 gene expression in primary cells and tumor cell lines.