(G) Immunoblotting analysis of whole-cell lysates (Input) and HA-immunoprecipitates (IP) obtained from HEK293T cells transfected with p100-DD (759900) alone or in combination with wild-type HA-ERK2. non-phosphorylated ERK2 and prevents ERK2 phosphorylation and nuclear translocation. Moreover, the death domain at C-terminal of p100 is identified as being crucial and sufficient for its interaction with ERK2. Taken together, our findings provide novel mechanistic insights into the understanding of the tumor suppressive role for NFB2 p100. Keywords: NFB2 (p100/ p52), PTEN, ERK2, miR-494, c-Jun == Introduction == Transcription factors of the nuclear factor B (NFB) play critical roles in a variety of biological and pathological processes. 1The NFB family consists of five members, NFB1 (p105/p50), NFB2 (p100/p52), RelA (p65), RelB, and c-Rel. 1Precursor protein NFB2 p100 is encoded bynfb2gene, and is well known as a fourth IB protein that suppresses both canonical and noncanonical NFB activation by preventing nuclear localization and DNA binding of NFB dimers. 2Genetic mutation or chromosomal rearrangements of thenfb2gene have been previously observed in human lymphomas and common variable immunodeficiency (CVID). 3, 4In addition, emerging evidence from The Cancer Genome Atlas (TCGA) has also revealed thatnfb2gene is genetically deleted or mutated in several human solid tumors including colorectal, gastric and prostate cancer, and that those colorectal cancer individuals with these alterations have poor clinical outcome, 5suggesting that NFB2 may play an inhibitory role in tumor development. Recently, the wild-type p100 has been reported to significantly inhibit tumor growth in severe combined immunodeficiency (SCID) mice, 6implicating p100 as a potential tumor suppressor. Although tumor suppressive effects of p100 have been well documented, the molecular mechanism underlying the anti-tumorigenic action of p100 remains poorly understood. PTEN (phosphatase and tensin homolog deleted on chromosome 10), a well-characterized tumor suppressor, 7principally acts as a negative regulator of PI3K/Akt signaling by dephosphorylating phosphatidylinositol-3, 4, 5-trisphosphate (PIP3), 8thus leading to inactivation of Akt and suppression of cell proliferation, cell survival and oncogenic cellular transformation. 7Despite frequent mutation or deletion ofPTENgene in human cancers, there are still 25% of cancer patients showing a positive correlation between loss ofPTENmRNA and its protein expression, 9indicating that the donwregulation of PTEN protein in those individuals Rabbit Polyclonal to SFRS4 could be attributed to the dysregulation of transcription factors involved in the regulation ofPTENtranscripts such as early growth-response protein 1 (EGR1)10and c-Jun11, as well as the non-coding RNAs that regulate the stability ofPTENmRNA includingPTENpseudogene 1 (PTENP1)12and microRNAs (miRNAs)9. NFB subunits, p65 and p50, have been previously reported to repressPTENtranscription through direct or indirect mechanisms. 13, 14However, as an inhibitory regulator of canonical and noncanonical NFB signaling, whether NFB2 has any regulatory roles in PTEN expression remains to be elucidated. Here, Angiotensin II we show that NFB2 p100 modulates PTEN expressionviaa mechanism that is independent of p100s inhibitory role in NFB signaling. Moreover, we identify that p100, but not p52, physically interacts with ERK2 and attenuates ERK2 phosphorylation, thereby leading to suppression of c-Jun/AP-1/miR-494 axis and stabilization ofPTENmRNA. == Results == == NFB2 deficiency promotes cancer cell anchorage-independent growth through PTEN inhibition == Although NFB subunits, p65 and p50, have been reported to repress PTEN expression at transcriptional level, 13, 14nothing is known about the roles of NFB2, p100 and p52, in the regulation of PTEN expression. To determine the regulatory roles of NFB2 in PTEN expression, we compared PTEN protein expression in NFB2+/+ and NFB2/ immortalized murine embryonic fibroblasts (MEFs). Intriguingly, NFB2 knockout led to a dramatic reduction of PTEN expression (Fig. 1A). Consistent with the modification of PTEN protein, Akt phosphorylation at Thr308/Ser473, a well-characterized PTEN downstream substrate, was markedly upregulated in NFB2/ cells (Fig. 1A). To define whether Angiotensin II these observed effects are the direct consequence of NFB2 deficiency, we used 2 units of specific short hairpin RNAs (shRNAs) targeting NFB2 to knockdown its expression in NFB2+/+ cells. We then established stable transfectants NFB2+/+(shNFB2-1#), NFB2+/+(shNFB2-2#), and their scramble control NFB2+/+(Nonsense) (Fig. 1B). The results obtained from these stable Angiotensin II transfectants consistently indicated that NFB2 inhibition impaired PTEN expression accompanied by an increase in Akt phosphorylation at Thr308/Ser473 (Fig. 1B). Due to frequent genetic deletion or mutation ofnfb2gene in human malignancies, 35we decided the biological roles of NFB2 in cancer cells by using human colon cancer HCT116 cells with wild-type NFB2 and wild-type PTEN. 1517Consistent with the observations in MEFs, knockdown of NFB2 expression in HCT116 showed a similar effect on PTEN expression and Akt phosphorylation (Fig. 1C). More importantly, soft-agar assay confirmed that NFB2 knockdown significantly promoted anchorage-independent growth of HCT116 cells (Fig. 1D), suggesting that NFB2 plays a suppressive role in cancer cellular transformation. To further investigate the association between the altered Angiotensin II PTEN expression and anchorage-independent growth in HCT116 cells upon NFB2 depletion, GFP-PTEN was re-introduced into.
(G) Immunoblotting analysis of whole-cell lysates (Input) and HA-immunoprecipitates (IP) obtained from HEK293T cells transfected with p100-DD (759900) alone or in combination with wild-type HA-ERK2
