Soluble adenylyl cyclase (sAC) was recently discovered as an independent source of cAMP besides the tranditional transmembrane adenylyl cyclase (tmAC). Compared to the tmAC, sAC is insensitive to G-protein and forskolin but is stimulated by bicarbonate and calcium ions. To better understand the function and regulation of sAC, our laboratory performed a yeast two-hybrid screen to identify its potential binding partners. We found that sAC has a physical interaction with another novel protein, p34
SEI-1. p34
SEI-1 is proposed to be localized in the nucleus and is regarded as a transcription regulator for E2F1/DP1 transcription activity.
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Soluble adenylyl cyclase (sAC) was recently discovered as an independent source of cAMP besides the tranditional transmembrane adenylyl cyclase (tmAC). Compared to the tmAC, sAC is insensitive to G-protein and forskolin but is stimulated by bicarbonate and calcium ions. To better understand the function and regulation of sAC, our laboratory performed a yeast two-hybrid screen to identify its potential binding partners. We found that sAC has a physical interaction with another novel protein, p34
SEI-1. p34
SEI-1 is proposed to be localized in the nucleus and is regarded as a transcription regulator for E2F1/DP1 transcription activity.
The sAC-p34
SEI-1 interaction was further confirmed by the glutathione S-transferase pull-down assay and co-immunoprecipitation. As recent studies suggested that p34
SEI-1 may also be localized in the cytoplasm, we carried a series of experiments to clarify this issue. Our subcellular fractionation and immunostaining experiments supported that p34
SEI-1 exists in the cytoplasm besides the nucleus. To study the functional sAC-p34
SEI-1 interaction, the effect of p34
SEI-1 expression on sAC generated cAMP was tested. Results showed that the sAC-mediated cAMP production significantly increased in the presence of p34
SEI-1. These data suggest that p34
SEI-1 acts as a novel positive regulator of sAC via physical interaction.
Also, the effect of their interaction on cAMP-response element-binding protein (CREB)-mediated transcription was investigated. Our luciferase assay using CRE as a promoter suggested that the sAC-p34
SEI-1 interaction may increase the CREB-mediated transcription activity in p34
SEI-1 stable cell-lines. This increase resulted from the sAC-p34
SEI-1 interaction instead of cAMP generated by sAC. However, the transiently expression of sAC and p34
SEI-1 suggests that p34
SEI-1 has an inhibitory effect on CREB-mediate transcription. We hypothesized that the high amount of p34
SEI-1 expressing in the cells may increase the p34
SEI-1 nuclear expression therefore generating inhibitory effect by binding to the coactivator of CREB, the CREB binding protein (CBP). A complete understanding of the effect of sAC-p34
SEI-1 interaction on CREB-mediated transcription still needs further investigation.
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