Endocytic organelles are organized as a transport network consisting of physically and biochemically distinct membranous domains, which serve as intracellular stations that integrate diverse signals to provide a high order of specificity and regulation to signaling. The end result of a signaling process is determined by factors, presumably associated with membrane vesicles that regulate the duration and intensity of the signal. Although it has long been presumed that receptorinitiated signaling at the plasma membrane is attenuated during internalization, few mechanistic details of the regulation are known. The family of NF-kappaB transcription factors plays an evolutionarily conserved and critical role in various biological processes such as proliferation, differentiation, immune response, and apoptosis. A number of diseases are caused by constitutive and dysregulation of NF-kappaB function including a wide variety of autoimmune diseases such as rheumatoid arthritis (RA), insulin-dependent diabetes mellitus, and multiple sclerosis, cancers, inflammatory diseases such as Crohns disease, ulcerative colitis, and arotic aneurysms. Inhibition of NF-kappaB activity as a treatment option is not viable because of the essentiality of its transcriptional function for normal immune response and cell survival. Thus knowledge of physiological pathways that tame or limit signal responsive NF-kappaB activation is of pressing clinical relevance. While plethora of different stress stimuli activates NF-kappaB, a detailed knowledge of the activation pathway is available only for limited number of stimuli including signaling pathways initiated by cytokines such as TNF. Usually receptor mediated signaling is turned off by ligand-bound receptor internalization and endocytosis. We hypothesized that proteins that are localized to intracellular compartments such as endocytic vesicles play essential role, under physiological conditions, in keeping the NF-kappaB transcription activation to optimal levels. Because of the obvious importance of knowledge of such pathways for treating many diseases we focused to investigate the role of proteins that regulate intracellular TNF signaling at membrane bound organelles. Our investigations have identified four proteins as novel regulators of TNF induced NF-kappB activation. Our investigation has led to the identification of caspase-8 and -10 associated RING proteins (CARPs; CARP-1 and CARP-2) as negative regulators of TNF-induced NF-kappaB activation. By virtue of their phospholipid-binding FYVE domain, CARPs localized to endocytic vesicles where it interacted with RIP in TNF-stimulated cells, resulting in RIP ubiquitination and degradation. Knockdown of CARPs stabilized TNFR1-associated ubiquitinated RIP levels after TNF simulation and enhanced activation of NF-kappaB. Therefore, CARPs acts at the level of endocytic vesicles to limit the intensity of TNF-induced NF-kappaB activation by the regulated elimination of a necessary signaling component within the receptor complex. Although both CARP1 and CARP2 exhibit similar function they are sequestered in distinct intracellular compartments suggesting that they may function in different endocytic pathways. Moreover, both these proteins exhibit different tissue patterns indicating that they function in cell-type specifc manner and/or play a role in other signaling pathways. Consistent with this idea we found that CARP-2 induces autophagy and the ubiquitin protein ligase (E3) activity is required for this function. Our data demonstrated that the expression of CARP2 in macrophage cell line elicits accumulation of autophagosomes as assessed by GFP-LC3 vacuole formation and conversion of endogenous LC3I form in to LC3II form and electron microscopy. Knockdown of CARP2 reduced starvation-induced autophagy. CARP2 appears to function downstream of mTOR but upstream of ATG5 and ATG7. We are presently working on the hypothesis hat CARP2-containing vesicles become part of the membrane around the autophagosomes. We have also generated knockout mice by deleting the entire portion of CARP1 and CARP2 separately and are breeding these knockout mice with wildtype black B6 mice to get homogenous genetic background. Thus far the homozygous deficient mice appear to be viable and healthy suggesting that the functional activity of these proteins is either not essential for animal development or each protein can compensate for the loss of activity of other protein.
| Fujita, Ken-Ichi; Srinivasula, Srinivasa (2009) Ubiquitination and TNFR1 Signaling. Results Probl Cell Differ : |
| Wu, Chuan-Jin; Conze, Dietrich B; Li, Tao et al. (2006) Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected] Nat Cell Biol 8:398-406 |
| Chao, Yang; Shiozaki, Eric N; Srinivasula, Srinivasa M et al. (2005) Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation. PLoS Biol 3:e183 |
| Saleh, Maya; Vaillancourt, John P; Graham, Rona K et al. (2004) Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms. Nature 429:75-9 |
| Maianski, N A; Geissler, J; Srinivasula, S M et al. (2004) Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis. Cell Death Differ 11:143-53 |
| Gupta, Sanjeev; Singh, Rajesh; Datta, Pinaki et al. (2004) The C-terminal tail of presenilin regulates Omi/HtrA2 protease activity. J Biol Chem 279:45844-54 |
| Yanamandra, Niranjan; Kondraganti, Shakuntala; Srinivasula, Srinivasa M et al. (2004) Activation of caspase-9 with irradiation inhibits invasion and angiogenesis in SNB19 human glioma cells. Oncogene 23:2339-46 |
