Senescence: DNA damage response triggers cytokine production

Severe genotoxic stress induces senescence and stimulates the DNA damage response pathway to induce secretion of inflammatory cytokines.

In response to DNA damage, the kinase ATM (ataxia telangiectasia mutated) phosphorylates multiple substrates to activate cell-cycle checkpoints and stimulate DNA repair. Damage that is not resolved causes persistent DNA-damage response (DDR) signaling, which leads to permanent p53-mediated growth arrest (senescence) and the development of a senescence-associated secretory phenotype (SASP). This phenotype is known to reinforce growth arrest in normal cells and promote cancer cell growth and invasion, but little is known about how the SASP is regulated. In Nature Cell Biology, Judith Campisi and colleagues now show that in senescent cells, persistent DDR signaling elicits secretion of the inflammatory cytokines interleukin (IL)-6 and IL-8 independent of p53 activity.

Severe DNA damage caused by ionizing radiation, expression of oncogenes or reactive oxygen species induced senescence, stable DDR signaling and the formation of persistent DNA damage foci (PDDF). Secretion of IL-6 and IL-8 – inflammatory cytokines involved in tumor promotion and key components of the SASP – increased after PDDF formation. In contrast, a low dose of ionizing radiation that caused transient DDR signaling, or senescence induced by the cyclin-dependent kinase inhibitor p16^INK4a, did not elicit PDDF formation or stimulate interleukin production, suggesting that development of the SASP depends on persistent DDR signaling.

Indeed, ATM depletion blocked IL-6 secretion, but not oncogene-induced PDDF formation or senescence. The DDR pathway proteins NBS1 and Chk2 were also required for establishing and maintaining IL-6 secretion by senescent cells with PDDF. However, neither p53 nor pRb was required for IL-6 secretion.

Conditioned media from senescent wild-type, but not ATM-deficient cells, stimulated the invasive capabilities of breast cancer cells in culture. Addition of exogenous IL-6 to the conditioned media from ATM-deficient cells rescued invasion, suggesting that DDR signaling induces a metastatic phenotype in trans. ATM depletion also blocked IL-6 secretion by premalignant cells, as well as cells undergoing oncogene-induced senescence. Furthermore, ATM substrate phosphorylation and IL-6 expression increased coincidentally in breast cancer specimens, suggesting that the DDR and inflammatory cytokine secretion are coupled in vivo.

Thus, severe genotoxic stress elicits PDDF formation and p53-regulated senescence, and also drives DDR-mediated secretion of inflammatory cytokines. A lingering question from this study is why DDR signaling elicits a SASP. The authors theorize that, in addition to the SASP acting in an autocrine manner to reinforce growth arrest, it might also signal in a paracrine manner to stimulate tissue repair or recruit immune cells to an area of damage. It will be important to explore these hypotheses in future studies.

Emily J. Chenette
Signaling Gateway

Reference:
Rodier, F. et al.
Persistent DNA damage signalling triggers s enescence-associated inflammatory cytokine secretion
Nature Cell Biology 11, 973-979 (2009)
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Further reading:
Fumagalli, M. and d'Adda di Fagagna, F.
SASPense and DDRama in cancer and ageing
Nature Cell Biology 11, 921-923 (2009)
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Kinases: Free ubiquitin!

The E3 ubiquitin ligase TRAF6 generates Lys63-linked unanchored polyubiquitin chains that bind to the TAK1 and IKK complexes to promote NF-κB activation.

The E3 ubiquitin ligase TRAF6 is known to activate NF-κB downstream of the interleukin-1 and Toll-like receptors. Together with the E2-type enzymes UBC13 and UEV1A, TRAF6 catalyzes Lys63-linked ubiquitination, which stimulates phosphorylation and activation of TAK1, consequent phosphorylation of the IκB kinase (IKK) and activation of NF-κB. In Nature, Zhijian Chen and colleagues now report on the mechanism of TRAF6-mediated NF-κB activation: TRAF6 generates unanchored polyubiquitin chains that bind to the regulatory subunits of the TAK1 and IKK complexes, leading to activation of these kinases.

To determine the requirement for TAK1 activation in vitro, purified TRAF6 was incubated with ubiquitin, an E1 enzyme, UBC13–UEV1A and the TAK1–TAB1–TAB2 kinase complex. TAK1 phosphorylation depended on the presence of UBC13–UEV1A, TRAF6 and ubiquitin with Lys at position 63. These data suggest that TRAF6-catalyzed Lys63-linked ubiquitination is necessary and sufficient for TAK1 activation. Intriguingly, when the in vitro assay was re-constituted without the TAK1–TAB1–TAB2 complex and then biochemically fractionated, the fraction of the reaction mixture without the ubiquitin ligase components was maximally able to activate TAK1. Further fractionation revealed that unanchored Lys63-linked polyubiquitin chains, but not monomeric ubiquitin, were able to activate TAK1 in a dose-dependent manner.

How do free Lys63-linked polyubiqutin chains lead to TAK1 activation? In IL-1β-stimulated cells, unanchored polyubiquitin chains were detected in association with TAB2 and NF-κB essential modulator (NEMO). Mutation of the ubiquitin-binding domains in TAB2 or NEMO blocked IKK activation. Furthermore, because an autophosphorylation-deficient mutant of TAK1 was nonetheless phosphorylated, the authors speculate that several TAB2 proteins might bind to a single polyubiquitin chain, bringing discrete TAK1 kinases into close proximity to facilitate their trans-phosphorylation. However, the possibility that ubiquitin binding promotes allosteric activation of the TAK1–TAB1–TAB2 complex cannot be ruled out.

Thus, TRAF6 activates NF-κB by generating free Lys63-linked polyubiquitin chains that bind and stimulate the TAK1 and IKK complexes. It will be interesting to discover whether this novel mechanism is utilized more widely in cellular processes.

Emily J. Chenette
Signaling Gateway

Reference:
Xia, Z.-P. et al.
Direct activation of protein kinases by unanchored polyubiquitin chains
Nature advance online publication, 12 August 2009 (DOI 10.1038/nature08247)
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GPCR subunits: Separate but not equal

A functional complementation assay reveals that maximal heterotrimeric G-protein activation is achieved by agonist binding to one subunit of a dopamine D2 receptor dimer.

What is the minimal functional unit of a G protein-coupled receptor (GPCR)? Some receptors have been proposed to exist in a 2:1 stoichiometry with heterotrimeric G proteins, although rhodopsin and the β₂ adrenergic receptor can activate G proteins in vitro as monomers. In addition, it is not always clear whether agonists bind one or both subunits of a receptor dimer. Reporting in Nature Chemical Biology, Jonathan Javitch and colleagues use a functional complementation assay to study the stoichiometry of human dopamine D2 receptor (D2R) signaling. They find that a D2R dimer binds to a single heterotrimeric G protein and is maximally activated by the binding of an agonist to one receptor protomer.

To study D2R activation, Han et al. fused one D2R (called protomer B) to a novel pertussis-toxin-insensitive G_qi5 chimera that produced calcium-dependent luminescence upon activation. The fusion protein did not signal in response to agonist binding because the very short linker did not allow the G protein to couple to the receptor to which it was fused. However, co-expression of a D2R protomer not fused to a G protein (called protomer A) caused robust agonist-mediated activation. Therefore, agonist-induced D2R signaling is mediated by two receptor protomers and one G protein.

A mutation in protomer A that inhibited agonist binding blocked G-protein-mediated signaling. Surprisingly, this same mutation in protomer B increased G-protein activation compared to the wild-type homodimer. Inverse agonist binding to protomer B also enhanced activation. Thus, the binding of an agonist to one subunit of a dimer is necessary and sufficient for G-protein activation. Intriguingly, agonist binding to protomer B, as well as a constitutively active version of protomer B, both diminished agonist-induced G-protein activation, suggesting that the active conformation of protomer B inhibits signaling and that agonist binding per se is not required for this effect.

Computational modeling and additional mutagenesis studies suggested that the second intracellular loop of both protomers makes contact with the G protein. However, the third intracellular loop of protomer A but not protomer B is required for G-protein activation, which indicates that each protomer in a receptor dimer has a discrete function. Given the apparent asymmetrical role of these protomers and the importance of conformational changes in modulating G-protein activation, it will be interesting to determine the effects of heterodimerization on agonist-stimulated signaling. The complementation assay described in this report will be a useful technique for these future studies.

Emily J. Chenette
Signaling Gateway

Reference:
Han, Y., Moreira, I. S., Urizar, E., Weinstein, H. and Javitch, J. A.
Allosteric communication between protomers of dopamine class A GPCR dimers modulates activation
Nature Chemical Biology advance online publication, 2 August 2009 (DOI 10.1038/nchembio.199)
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Oncogenesis: The many faces of C-CBL

Mutations in the E3 ubiquitin ligase C-CBL that unmask its oncogenic potential have been detected in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are a group of blood cancers that can progress to acute myeloid leukemia (AML). In Nature, Seishi Ogawa and colleagues now report that C-CBL, an E3 ubiquitin ligase proposed to function as a tumor suppressor, is mutated in MDS/CMML patients. Surprisingly, mutant C-CBL promotes oncogenic transformation of immature hematopoietic stem and progenitor cells (HSPCs), suggesting that C-CBL possesses both tumor suppressor and proto-oncogenic capabilities.

Single nucleotide polymorphism (SNP) arrays detected a loss of heterozygosity associated with acquired uniparental disomy in 17 MDS samples in a chromosomal region containing the C-CBL locus. Mutations in C-CBL were detected in 15 of the samples; of these, most samples were from patients with CMML. These mutations clustered in the C-CBL linker–RING-finger domain and, based on the crystal structure of C-CBL, were expected to block ubiquitin ligase activity. Indeed, whereas co-expression of wild-type C-CBL with a variety of tyrosine kinases induced their ligand-dependent ubiquitination, expression of mutant C-CBL blocked ubiquitination, leading to prolonged activation of the kinases.

The loss of C-CBL in HSPCs increased cellular proliferation in response to cytokines. Intriguingly, expression of mutant C-CBL in the knockout cells markedly enhanced their proliferative response to a wider variety of cytokines, likely via the PI(3)K–Akt and STAT5 signaling pathways. Co-expression of wild-type C-CBL blunted this response, revealing the importance of loss of heterozygosity in unmasking C-CBL's oncogenic potential.

These findings indicate that the C-CBL mutations seen in MDS/CMML patients represent gain-of-function mutations. Deregulation of tyrosine kinase and Ras signaling pathways has been implicated in the pathogenesis of MDS/CMML, and C-CBL mutations represent a previously unappreciated mechanism by which these pathways may be upregulated. However, the precise molecular mechanism by which mutant C-CBL stimulates cytokine-induced proliferation and oncogenic transformation is not yet known. As persistent tyrosine kinase signaling is a hallmark of many types of cancer, it will be important to determine whether C-CBL is mutated in other cancers.

Emily J. Chenette
Signaling Gateway

Reference:
Sanada, M., Suzuki, T., Shih, L.-Y. et al.
Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms
Nature advance online publication, 20 July 2009 (DOI 10.1038/nature08240)
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