Roles of specific isoforms of protein kinase G in downregulation of cyclin D1 and growth inhibition

초록

Protein kinase G (PKG) has been implicated in induction of apoptosis and growth inhibition. To elucidate the roles of specific PKG isoforms in the transcription control of growth control genes, we constructed a series of expression vectors of PKG1-a and PKG1-b which encode HA-tagged wild type, constitutively active and dominant negative mutants and examined the effects of these constructs on the transcription of cyclin D1 and other cell cycle genes. Our present study demonstrates that the constitutively active mutants of PKG1-b downregulate the transcription of cyclin D1 when transiently transfected in NIH3T3 cells, while PKG1-a mutants show no significant effect. We then examined the effects of PKG constructs on several enhancer elements found in cyclin D1 promoter, such as, c-fos, c-jun, SRE and TRE using the luciferase reporter assay. Constitutively active mutants of PKG1-b showed marked transcriptional downregulation of c-fos in NIH3T3 cells, whereas PKG1-a showed lesser extent. We also found that the constitutively active mutants of PKG1-b inhibit the activation of SRE and TRE, suggesting their involvement in the regulation of cyclin D1. Interestingly we did not observe any negative effect on SRE or TRE by PKG1-a mutants. These studies indicate that the activation of PKG1-b isoform in mammalian cells may be responsible for the downregulation of cyclin D1 and implicate a novel anti-proliferative signaling pathway.