Investigation of the role of protein kinase-a in E2F1transcription factor activation

E2F1 gene, which was discovered by the demonstration that adenoviral E1 and E2 genes were responsible for transcription of viral genome, resides on the long arm of 20th chromosome. Its product is a protein comprising 437 aminoacids. After dimerizing with its partner, E2F1 becomes active during G1 phase of cell cycle and induces the transcription of numerous cell cyle-associated genes such as that of DNA Polymerase. As well as being transcriptionally controlled, E2F1 activity is modulated by means of post-translational modifications. Post-translationally, E2F1 is acetylated by P/CAF, methylated by Set 9 and phosphorylated by CHK2. While Set 9-mediated methylation leads to degradation of E2F1, CHK2 and P/CAF-mediated modifications prevent Set9-mediated methylation and prevents its degradation. In addition to post-translational modifications occurring in response to pathological situations like UV exposure and DNA damage, E2F1 can also undergo post- translational modifications by various molecules under physiological conditions. Epinefrin, adrenalin, endotellin-1 and numerous chemokines play pivotal role in cell physiology, and these transmit their signals by binding to GPCR (G-protein coupled receptors). Activation of these receptors by above mentioned ligands induces activation of several pathways one of which is Adenylate cyclase. Activated adenylate cyclase produces cAMP from ATP. In addition to these, cellular cAMP levels is also increased when glucose level goes down. cAMP is the first second messenger discovered and elicits its biological effect by activating PKA enzyme. Activated PKA elicits its biological effect by phosphorylating target proteins which contain Serin or Treonin amino acids in concensus RXXS/T motif. In general, increase of cellular cAMP level and resulting PKA activation negatively regulate cell proliferation. Under the light of these we thought that activated PKA would negatively affect the activity of E2F1 which controls initiation of cell cycle. In this regard, to see whether E2F1 can be a substrate for PKA we looked at the amino acid sequence of E2F1 and found three hypothetical consensus PKA phosphorylation sites. These phosphorylation sites are RYET, RLLS ve RMGS amino acid sequences and these are localized at 127-130, 232-235 ve 361-364 positions. By assuming the fact that Serin and Treonin at these sites will be phosphorylated by PKA, and these phosphorylations would affect the activity of E2F1, we converted codons of Serine and Treonin to non-phosphorylatable Alanin and phosphorylation mimicking Glutamic acid codon on eukaryotic E2F1 expression vector we created. According to our results, PKA-mediated phosphorylation of E2F1 seems to have an effect on cell proliferation, induction of apoptosis, glucose uptake and cisplatin-induced cell death.