Usha Ponnappan

Department of Geriatrics, Medicine, Microbiology and Immunology, University of Arkansas for Medical Sciences, and GRECC, John L. McClellan Memorial VA hospital, VA Medical Research, GC143, 151/LR, 4300 West 7^th street, Little Rock, Arkansas 72205

Received 1/15/97 Accepted 1/29/98

3. TRANSCRIPTION FACTOR NF KAPPA B

NF kappa B is induced in response to many different noxious agents. It is activated by a myriad of agents including cytokines like IL-1 and TNF-alpha, bacterial LPS, Viral infection and certain viral proteins such as HTLV-1 Tax, LMP1 of EBV, antigen receptor cross linking on T and B lymphocytes, Calcium ionophores, phorbol esters, UV radiation and others. The genes regulated by NF kappa B family of transcription factors are just as diverse as the activators and include those involved in immune function, inflammatory response, cell adhesion, cell growth, and cell death. First characterized in mature B and plasma cells as a nuclear protein that binds specifically to a 10-bp sequence in the kappa intronic enhancer , NF kappa B has now been demonstrated in virtually all cells. In most cells with the exception of mature B cells, macrophages and some neurons, NF kappa B remains dormant in the cytoplasm bound to its inhibitor, I kappa B. Treatment with various agents leads to the dissociation of the inhibitor and the translocation of the free NF kappa B to the nucleus.

NF kappa B was originally identified as a B lymphocyte nuclear factor binding to a site in the immunoglobulin kappa light chain enhancer (85). It is now recognized as a pleiotropic transcription factor binding to many cellular and viral gene promoters. NF kappa B is recognized for its central role in immunological processes via the expression of a wide variety of immune response genes (86-89). Recent studies have provided evidence for the involvement of NF kappa B in growth control of certain tumors (90-94). NF kappa B is maintained in the cytoplasm of the cell by the inhibitor kappa B (I kappa B) which tightly regulates the nuclear expression and biological function of NF kappa B (95-97). The speed of induction and its ubiquitous expression makes NF kappa B an ideal regulator of rapid-response genes (87,98). NF kappa B family of transcription factors are homo- and heterodimeric complexes formed from combinations of members of the Rel family of proteins (86-89). The Rel family of proteins belong to the v-rel oncogene found in the Reticuloendotheliosis Virus Strain T (RevT) and are characterized by having a common Rel homology domain (RHD) which consists of 300 amino acids in length. Specific sites within the RHD are responsible for DNA binding to kappa B sites, dimerization with other Rel family proteins, and interaction with I kappa B. The C-terminal portion of the RHD contains a group of positively charged amino acids that function as the nuclear localization signal (NLS) (86-89, 99,100). There are five mammalian members of the Rel family of proteins. These include c-Rel, NF kappa B1 (p105/p50), NF kappa B2 (p100/p52), RelA (p65), and RelB (86-89). NFATp(nuclear factor of activated T cells) is considered to be related to the Rel family of proteins as it has a region of 430 amino acids that shares 17% amino acid identity to RelA (p65). There also appears to be some functional similarities between p65 and NFATp since both these proteins can interact with c-fos and c-jun (101-103).

Theoretically, five members of the Rel family proteins can form almost any possible combination of homo or heterodimers although only certain combinations have been detected in vivo (89).The classic and most well studied NF kappa B molecule is a heterodimer of p50/p65 subunits. This heterodimer is the most abundant complex and is found in virtually all cell types. Heterodimers of p50/p65 are rapidly translocated to the nucleus following cellular activation and bind the consensus sequence 5'GGGRNNYYCC3'. The transactivation function in vivo is mediated by RelA (p65), RelB, and c-Rel which contain transactivation domains in their C-terminal domains, while the p50 and p52 subunits primarily serve as DNA binding subunits (86,104-108). Transcriptionally active complexes are usually heterodimers consisting of p50 or p52 in combination with one of the transactivating subunits (p65, c-Rel, or RelB), while homodimers of p50 correlate with transcriptional repression (86,109,110). Each of the heterodimers exhibit unique properties including cell type specificity, DNA binding site preference, differential interactions with I kappa B isoforms, differential activation requirements, and kinetics of activation, thus being capable of regulating gene expression in a uniquely specific manner (86,104,111-115). For example, in pre-B cells the active NF kappa B complexes are primarily dimers of p50/p65 while the constitutive form of NF kappa B in mature B cells are c-Rel/p50 dimers (114). While p50/p65 dimers rapidly appear in the nucleus following stimulation, dimers of p50/c-Rel exhibit a more delayed response and accumulate in the nucleus more slowly (115).

3.1 Inhibitor of kappa B, I kappa B

The rapid inducibility of NF kappa B can be attributed to the fact that it preexists in the cytoplasm of cells in an inactive form complexed to I kappa B, thus requiring no new protein synthesis (96-98). I kappa B proteins regulate the cellular location, DNA binding, and transcriptional properties of NF kappa B/Rel family of proteins. The current mammalian I kappa B family of proteins includes I kappa B-alpha, I kappa B-beta, I kappa B-gamma, I kappa B-delta, (generated from alternative splicing of the NF kappa B1 gene, NF kappa B1, NF kappa B2 ), I kappa B-epsilon and the predominantly nuclear protein Bcl-3 (86,87,116). All I kappa B proteins have in common a conserved domain containing six to eight repeats of the erythrocyte protein ankyrin (117,118). I kappa B binds to the NF kappa B dimer and masks its nuclear localization signal thereby sequestering NF kappa B in the cytoplasm of the cell (95). The NF kappa B/I kappa B complex itself cannot bind to DNA, however disassociation of I- kappa B from NF kappa B which can be achieved in vivo with various activating agents or in vitro with agents such as deoxycholate will produce an NF kappa B dimer which is capable of translocating to the nucleus and binding DNA (96,119).

I kappa B-alpha is the most extensively studied and most abundant I kappa B family member and unlike I kappa B-beta, is primarily involved in regulating the rapid and transient activation of NF kappa B (116,120). I kappa B-alpha is a 37 kd protein which can be structurally divided into a 70 amino acid N terminus, a central section of 205 amino acids composed of 6 ankyrin repeats, and an acidic 42 amino acid C- terminus that contains a PEST (pro-glu-ser-thr) sequence, a motif correlated with rapid protein turnover. I kappa B-alpha performs several critical functions including cytoplasmic retention of NF kappa B in resting cells, release of NF kappa B in response to activating signals, and inhibition of DNA binding by NF-kappa B (87,121). I kappa B-alpha binds to specific Rel subunits and masks the NLS of all dimers containing any of the transactivating subunits (RelA, c-Rel, RelB), especially those containing RelA, thereby retaining these complexes in the cytoplasm (87). Studies of stoichiometry have shown that one dimer of NF kappa B is bound to one I kappa B-alpha molecule (118,121). Thus the classic cytoplasmic NF KAPPA B complex contains a p50/p65 dimer bound to one I kappa B-alpha. I kappa B-alpha can have differential affinity for the various NF kappa B dimers. For example, I kappa B-alpha binds a RelB/p50 heterodimer more efficiently than a RelB/p52 heterodimer. Complexes with the highest affinity for I kappa B-alpha are thought to be mainly cytoplasmic and represent the inducible pool, while those complexes with low affinity for I kappa B-alpha are nuclear and provide constitutive activity.

Removing DNA bound dimers in the absence of an activating signal may also be a role of I kappa B-alpha since I kappa B-alpha not only prevents DNA binding of strongly activating complexes but can also dissociate bound complexes from DNA (122). The underlying mechanism for the inhibition of DNA binding mediated via I kappa B-alpha is not clearly understood, however, it has been shown to require the C-terminal region of I kappa B-alpha (121). Recent studies have shown that exogenously introduced, over expressed, or newly synthesized I kappa B-alpha can be found not only in the cytoplasm, but in the nucleus as well (123-125). In addition, recent studies of I kappa B-alpha knockout mice have demonstrated that TNF-alpha treatment of embryonic fibroblasts from these mice results in a prolonged and sustained nuclear induction of NF kappa B indicating that I-kappa B-alpha plays a role in the termination of an NF kappa B response (126). Thus, it is likely that newly synthesized I kappa B-alpha may enter the nucleus and regulate NF kappa B activity, resulting in a transient response.