The Nm23 metastasis suppressor family is involved in diverse physiological and pathological processes including tumorigenesis and metastasis. Although the inverse correlation of Nm23 level with tumor metastasis potential has been widely observed, the mechanisms that regulate the expression of Nm23 remain poorly understood. Previous studies have revealed that Nm23-H1/2 isoforms are upregulated by RGS19, a regulator of G protein signaling (RGS) protein which accelerates the termination of G_i signals. Here, I examined the transcriptional regulation of Nm23-H1/2 by in silico analysis and luciferase assays. Both transient and stable overexpression of RGS19 upregulated the Nm23-H1/2 protein levels and activated several transcription factors including CREB, AP-1 and SRE in HEK293 cells. Elevat...[ Read more ]

The Nm23 metastasis suppressor family is involved in diverse physiological and pathological processes including tumorigenesis and metastasis. Although the inverse correlation of Nm23 level with tumor metastasis potential has been widely observed, the mechanisms that regulate the expression of Nm23 remain poorly understood. Previous studies have revealed that Nm23-H1/2 isoforms are upregulated by RGS19, a regulator of G protein signaling (RGS) protein which accelerates the termination of G_i signals. Here, I examined the transcriptional regulation of Nm23-H1/2 by in silico analysis and luciferase assays. Both transient and stable overexpression of RGS19 upregulated the Nm23-H1/2 protein levels and activated several transcription factors including CREB, AP-1 and SRE in HEK293 cells. Elevation of intracellular cAMP level upregulated the expression of Nm23-H1/2 via the activation of protein kinase A (PKA) but not exchange proteins activated by cAMP (EPAC) in both non-cancer cells and metastatic cancer cell lines. Conversely, inhibition of protein kinase A (PKA) suppressed the phosphorylation of CREB and reduced the expression of Nm23-H1/2. Since Nm23 as metastasis suppressor is capable of inhibiting cancer cell migration, the effect of PKA activation on cancer cell migration was examined. Transwell assay and wound healing assay indicated that the activation of cAMP/PKA suppresses the migration of cancer cells. These results suggest a novel regulatory route to manipulate metastasis suppressor Nm23 in a cAMP/PKA/CREB-dependent mechanism, which provides a mechanism in understanding the regulation of metastasis by PKA pathway. On the other hand, the two most studied members, Nm23-H1 and H2 have hexameric structures formed by the combination of dimers and can interact with different protein partners. To understand how the hexamers form from dimers and to identify the structural determinants for this assembly process, mutations were constructed and the characteristics of the mutants were examined. For two clusters located inside Nm23-H1 dimers, different combination of residues substituted by alanine differentially affected the protein expression levels, suggesting that these residues play important roles in facilitating protein synthesis or maintaining protein stability. For the surface mutants of Nm23-H1, seven mutants with double or triple mutations have been produced by swapping the differential sites of Nm23-H1 and H2 on exposed molecular surfaces. Mutants H1^KS and H1^KSK might change the protein post-translational modification towards Nm23-H2, since there is obvious mobility shifts in electrophoresis for these two mutants. Besides, co-immunoprecipitation assays indicate that both Nm23-H1 and H2 bind to Gβγ dimer, but association with Gβγ was also observed in two of the mutants (H1^KHD and H1^HD). Collectively, my results suggest that Nm23-H1 has specific structural characteristics in defining hexamer formation as well as protein-protein interactions. Importantly, a PKA-dependent mechanism for controlling Nm23-H1/2 expression is proposed, which may provide new approaches to limit cancer metastasis by modulating the expression of the Nm23 metastasis suppressors.