Nuclear TRAF3 is a negative regulator of CREB in B cells Nurbek Mambetsarieva,b,c,1, Wai W. Linb,1, Laura L. Stunza,d, Brett M. Hansona, Joanne M. Hildebranda,2, and Gail A. Bishopa,b,d,e,f,3 aDepartment of Microbiology, University of Iowa, Iowa City, IA 52242; bImmunology Graduate Program, University of Iowa, Iowa City, IA 52242; cMedical Scientist Training Program, University of Iowa, Iowa City, IA 52242; dHolden Comprehensive Cancer Center, University of Iowa, Iowa City, IA 52242; eInternal Medicine, University of Iowa, Iowa City, IA 52242; and fDepartment of Veterans Affairs Medical Center, Research (151), Iowa City, IA 52246 Edited by Louis M. Staudt, National Cancer Institute, NIH, Bethesda, MD, and approved December 14, 2015 (received for review July 23, 2015) The adaptor protein TNF receptor-associated factor 3 (TRAF3) regu- deficient T cells and macrophages lack the enhanced survival phe- lates signaling through B-lymphocyte receptors, including CD40, notype, although they display constitutive NF-κB2 activation (12, BAFF receptor, and Toll-like receptors, and also plays a critical role 13). TRAF3 degradation is neither necessary nor sufficient for B-cell inhibiting B-cell homoeostatic survival. Consistent withthesefindings, NF-κB2 activation (14). These findings indicate that TRAF3 regu- loss-of-function human TRAF3 mutations are common in B-cell cancers, lates additional important prosurvival pathways in B cells. particularly multiple myeloma and B-cell lymphoma. B cells of B-cell– Nuclear localization of TRAF3 has been reported in several specific TRAF3−/− mice (B-Traf3−/−) display remarkably enhanced sur- nonhematopoietic cell types (15, 16), but the function of TRAF3 vival compared with littermate control (WT) B cells. The mechanism in the nucleus is poorly understood. Pathway analysis of prelimi- for this abnormal homeostatic survival is poorly understood, a key nary microarray gene expression data comparing B cells isolated − − knowledge gap in selecting optimal treatments for human B-cell from WT (littermate control) and B-Traf3 / mice identified cAMP cancers with TRAF3 deficiency. We show here for the first time to response element binding protein (CREB) as a transcription factor our knowledge that TRAF3 is a resident nuclear protein that asso- potentially dysregulated in the absence of TRAF3 specifically in B − − ciates with the transcriptional regulator cAMP response element cells, as this pathway was not altered in TRAF3 / T cells. binding protein (CREB) in both mouse and human B cells. The CREB is a ubiquitous transcription factor implicated in sur- TRAF-C domain of TRAF3 was necessary and sufficient to localize vival and proliferation of immune cells (17). CREB has also been TRAF3 to the nucleus via a functional nuclear localization signal. suggested to promote BAFF-mediated B-cell survival, via its − − CREB protein was elevated in TRAF3 / B cells, without change in induction of Mcl-1, an antiapoptotic member of the Bcl-2 family, mRNA, but with a decrease in CREB ubiquitination. CREB-mediated but the mechanism of this induction is unknown (10). In this transcriptional activity was increased in TRAF3-deficient B cells. Con- study, we show that TRAF3 is a resident nuclear protein in B cells, sistent with these findings, Mcl-1, an antiapoptotic target of CREB- and that nuclear, but not cytoplasmic TRAF3 interacts with mediated transcription, was increased in the absence of TRAF3 CREB to inhibit CREB stability and CREB-mediated transcrip- and enhanced Mcl-1 was suppressed with CREB inhibition. TRAF3- tion. An important CREB-regulated transcriptional target, Mcl-1, deficient B cells were also preferentially sensitive to survival inhibi- is up-regulated in TRAF3-deficient B cells. These findings have tion with pharmacologic CREB inhibitor. Our results identify a new important implications for both the regulation of homeostatic mechanism by which nuclear TRAF3 regulates B-cell survival via in- B-cell survival, as well as the pathogenesis of B-cell malignancies hibition of CREB stability, information highly relevant to the role of that harbor TRAF3 deficiency. TRAF3 in B-cell malignancies. Significance Bcell| CREB | TRAF3 Uniquely in B cells, deletion of the adaptor protein TRAF3 (TNF RAF3 (TNF receptor-associated factor 3) is an important receptor-associated factor 3) causes enhanced survival; TRAF3 Tmember of the TRAF adaptor family with distinct cell and deficiency is observed in a substantial percentage of human context-specific roles (1). Our group previously generated B-cell malignancies. Here, we report that TRAF3 is a resident Cre flox/flox −/− CD19 TRAF3 mice (B-Traf3 ) that lack TRAF3 spe- nuclear protein in B cells that regulates stability of the CREB cifically in B cells. This deletion results in remarkably enhanced (cAMP response element binding protein) transcription factor. − − B-cell survival in vitro and in vivo (2). Aged B-Traf3 / mice have This regulation is important for restraining B-cell survival by substantially higher incidence of spontaneous B-cell lymphoma, preventing transcription of prosurvival genes and may be a key further supporting the tumor-suppressive role of TRAF3 in B cells mechanism by which loss of TRAF3 contributes to B-cell malig- (3). Studies of human tumors identified loss-of-function TRAF3 nancies. Considering the high frequency of TRAF3 mutations in mutations in nearly 20% of multiple myeloma (MM); TRAF3 is B-cell tumors, our findings of an important nuclear role for now recognized as one of the top 11 genes mutated in two-thirds TRAF3 present a new paradigm in understanding both normal of MM tumors (4). Additionally, ≥15% of diffuse large B-cell regulation of B-cell survival and pathogenesis of B-cell cancers. lymphomas are now known to harbor TRAF3 mutations (5, 6). Lesions in human TRAF3 genes are also seen in Hodgkin’slym- Author contributions: N.M., W.W.L., L.L.S., and G.A.B. designed research; N.M., W.W.L., L.L.S., B.M.H., and J.M.H. performed research; J.M.H. contributed new reagents/analytic phoma (7) and associated with particular chromosome 14 deletions tools; N.M., W.W.L., L.L.S., B.M.H., and G.A.B. analyzed data; and N.M., W.W.L., and G.A.B. in various B-cell lymphomas (8). Interestingly, Traf3 mutations are wrote the paper. also common in canine B-cell lymphomas (5). The authors declare no conflict of interest. κ TRAF3 is a negative regulator of the noncanonical NF- B(NF- This article is a PNAS Direct Submission. κ B2) pathway, and enhanced survival in TRAF3-deficient B cells is 1N.M. and W.W.L. contributed equally to this work. κ associated with constitutive activation of NF- B2 (2, 9). BAFF binds 2Present address: Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, to BAFF receptor (BAFFR) to activate a complex signaling cascade Australia. that includes TRAF3 degradation and NF-κB2 activation, ultimately 3To whom correspondence should be addressed. Email: [email protected]. κ promoting B-cell survival (10, 11). However, NF- B2 activation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. is not sufficient to promote enhanced survival, because TRAF3- 1073/pnas.1514586113/-/DCSupplemental. 1032–1037 | PNAS | January 26, 2016 | vol. 113 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1514586113 Downloaded by guest on September 30, 2021 Results lacking the TRAF-C domain, was identified in the LP1 MM cell Association of Nuclear TRAF3 with CREB in B Cells. In search of novel line (LP1 mutant) (24) (Fig. S1A). When stably and inducibly −/− targets of TRAF3 that contribute to its B-cell–unique function in expressed by transfection in TRAF3 CH12.LX B cells, LP1 regulating homeostatic survival, we identified CREB as a tran- promotes elevated NF-κB2 activation (14). Interestingly, LP1 scription factor pathway, dysregulated specifically in B cells in failed to localize to the nucleus or associate with CREB (Fig. 2A). the absence of TRAF3. In B cells, CREB is activated in response Furthermore, the TRAF-C domain alone was sufficient for nu- to B-cell antigen receptor (BCR) (18) or BAFFR stimulation, clear localization (Fig. 2B). To confirm the role of the NLS in possibly downstream of the kinase Akt (10). Enhanced B-cell targeting TRAF3 to the nucleus, we generated a point mutant survival resulting from absence of TRAF3 is BAFF independent (ΔNLS) that has two lysines replaced with alanines in the NLS (2), and it was recently reported that TRAF3 is an important sequence (Fig. S1B). The ΔNLS mutant showed markedly inhibited regulator of transcription factor degradation (19). We thus hy- capacity for nuclear localization (Fig. 2C). These findings demon- pothesized that TRAF3 regulates CREB in B cells to restrain strate that the NLS in the TRAF-C domain of TRAF3 is critical their homeostatic survival. for its trafficking to the nucleus. The presence of truncated TRAF3 To investigate a potential association between TRAF3 and mutants lacking that domain in human B-cell malignancies (24) CREB, we immunoprecipitated CREB and its coactivator CREB- strongly suggests that loss of nuclear TRAF3 may specifically binding protein (CBP) from cytoplasmic and nuclear lysates of contribute to disease pathogenesis. + + − − parental (TRAF3 / ) and TRAF3 / subclones of the CH12.LX mouse B-cell lymphoma line (20). Both CREB and CBP specifi- TRAF3-Mediated Negative Regulation of CREB. TRAF3 negatively cally associated with TRAF3 in parental cells, but the association regulates stability of certain transcription factors in macrophages was seen almost exclusively in the nucleus and not the cytoplasm (19). We thus hypothesized that TRAF3 interaction with CREB (Fig. 1 A and B). regulates CREB nuclear stability in B cells. B cells isolated from −/− −/− We confirmed nuclear localization of TRAF3 in mouse pri- B-Traf3 (TRAF3 ) mice had increased levels of both total mary B cells via both cellular fractionation and in intact cells, and phosphorylated cellular CREB protein (Fig.