Biomedical subjects

Parameswaran Ramakrishnan

Publications and source records attributed to Parameswaran Ramakrishnan.

5 recordsLinked to original sources

PubMed2006

NF-kappaB-inducing kinase is involved in the activation of the CD28 responsive element through phosphorylation of c-Rel and regulation of its transactivating activity.

Previous evidence suggested that NF-kappaB-inducing kinase (NIK) might regulate IL-2 synthesis. However, the molecular mechanism is not understood. In this study, we show that NIK is involved in CD3 plus CD28 activation of IL-2 transcription. Splenic T cells from aly/aly mice (that have a defective NIK protein) have a severe impairment in IL-2 and GM-CSF but not TNF secretion in response to CD3/CD28. This effect takes place at the transcriptional level as overexpression of alyNIK inhibits IL-2 promoter transcription. NIK activates the CD28 responsive element (CD28RE) of the IL-2 promoter and strongly synergizes with c-Rel in this activity. We found that NIK interacts with the N-terminal domain of c-Rel, mapping this interaction to aa 771-947 of NIK. Moreover, NIK phosphorylates the c-Rel C-terminal transactivation domain (TAD) and induces Gal4-c-Rel-transactivating activity. Anti-CD28 activated Gal4-c-Rel transactivation activity, and this effect was inhibited by a NIK-defective mutant. Deletion studies mapped the region of c-Rel responsive to NIK in aa 456-540. Mutation of several serines, including Ser471, in the TAD of c-Rel abrogated the NIK-enhancing activity of its transactivating activity. Interestingly, a Jurkat mutant cell line that expresses one of the mutations of c-Rel (Ser471Asn) has a severe defect in IL-2 and CD28RE-dependent transcription in response to CD3/CD28 or to NIK. Our results support that NIK may be controlling CD28RE-dependent transcription and T cell activation by modulating c-Rel phosphorylation of the TAD. This leads to more efficient transactivation of genes which are dependent on CD28RE sites where c-Rel binds such as the IL-2 promoter.

Amino Acid Sequence↗

PubMed2006

Hsp90 recognizes a common surface on client kinases.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Amino Acid Sequence↗

PubMed2005

TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs.

TNF family members and their receptors contribute to increased gene expression for inflammatory processes and intracellular cascades leading to programmed cell death, both via activation of NF-kappaB. TNF receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins binding to various receptors of the TNFR family. In an attempt to delineate the role of individual TRAFs, we compared NF-kappaB activation by CD40(wt) and CD40 mutants with different TRAF recruitment patterns. Recognized only recently, NF-kappaB signaling occurs at least via two different pathways. Each pathway results in nuclear translocation of two different Reldimers, the canonical p50/RelA and the noncanonical p52/RelB. Here, we show that via TRAF6, CD40 mediates only the activation of the canonical NF-kappaB pathway. Via TRAF2/5, CD40 activates both the canonical and the noncanonical NF-kappaB pathways. We observed that TRAF3 specifically blocked the NF-kappaB activation via TRAF2/5. This inhibitory effect of TRAF3 depends on the presence of an intact zinc finger domain. Paradoxically, suppression of TRAF2/5-mediated NF-kappaB activation by TRAF3 resulted in enhanced transcriptional activity of TRAF6-mediated canonical NF-kappaB emanating from CD40. We also observed that 12 TNFR family members (p75TNFR, LTbetaR, RANK, HVEM, CD40, CD30, CD27, 4-1BB, GITR, BCMA, OX40, and TACI) are each capable of activating the alternative NF-kappaB pathway and conclude that TRAF3 serves as a negative regulator of this pathway for all tested receptors.

CD40 Antigens↗

PubMed2004

Caspase-8 serves both apoptotic and nonapoptotic roles.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

Animals↗

PubMed2004

Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase.

The NF-kappaB-inducing kinase (NIK) induces proteolytic processing of NF-kappaB2/p100 and, hence, the generation of NF-kappaB dimers such as p52:RelB but was suggested not to signal for the processing of IkappaB. Here, we show that although the induction of IkappaB degradation in lymphocytes by TNF is independent of NIK, its induction by CD70, CD40 ligand, and BLyS/BAFF, which all also induce NF-kappaB2/p100 processing, does depend on NIK function. Both CD70 and TNF induce recruitment of the IKK kinase complex to their receptors. In the case of CD70, but not TNF, this process is associated with NIK recruitment and is followed by prolonged receptor association of just IKK1 and NIK. Recruitment of the IKK complex to CD27, but not that of NIK, depends on NIK kinase function. Our findings indicate that NIK participates in a unique set of proximal signaling events initiated by specific inducers, which activate both canonical and noncanonical NF-kappaB dimers.

Antigens, CD↗