CT Ig-like Domain NT Ig-like I Domain ' W C NLS y RHD
F igu re 1-8: Schem atic R epresen tation o f the lK B /p 6 5 /p 5 0 C rystal Structure (red raw n from B aeuerle, 1998)
(B aeu erle, 1998). Shown in purple is k B a , with its 6 ankyrin repeats. Shown in green is p50 and in blue p65. Each
contains tw o Ig-like domains. Dashed lines indicate sequences m issing from the structures. RHD = Rel H omology
Domain; P = phosphate group. The structure was com posed com bining features from tw o independent studies
(Huxford et ai, 1998; Jacobs and Harrison. 1998).
H etero trim eric c ry sta ls c o n ta in in g th e C -term in a l Ig -lik e d o m a in o f th e p 5 0 R H D , th e en tire R H D o f p 6 5
an d a tru n cated v e rs io n o f th e I x B a p rotein w e r e o b ta in e d . T h e stru ctu res p r e se n ted b y both g r o u p s la ck ed
th e N -te rm in a l R H D o f p 5 0 and la ck ed th e m a jo rity o f th e N - and C -reg u la to ry d o m a in s o f I k B a .
N e v e r th e le s s , th e s e stu d ies r e v e a le d that th e an k yrin rep ea ts in I x B a form a slig h tly b en t c y lin d er w ith 5
lo o p s p rotru d in g from th e sta ck ed a - h e lic e s , w h ic h co n ta in r e s id u e s r e s p o n s ib le for r e c o g n itio n o f N FkB.
T h e in teraction o f 1kB « w ith p 5 0 /p 6 5 in v o lv e d a ll s ix an k yrin rep ea ts, w ith rep ea ts 3 -5 o f I x B a m a k in g th e
It remains unclear as to whether this interaction disrupts the binding of NFkB to DNA, but ankyrin repeat
6
and part of the PEST sequence of IkBq were shown to bind to residues within the RHD of p65 and this is thought to have the largest impact on DNA binding (Huxford et a l, 1998). When complexed to the IkB inhibitor, the N-terminal Ig-like domain of the p65 RHD is rotated by 180° compared to the situation with the DNA-bound complex, occluding critical residues required for DNA contact. Jacobs and Harrison also showed that ankyrin repeats 1 and 2 force the NLS and flanking sequences of p65 into an a-helical confirmation that may not be compatible for recognition of the NLS by nuclear transport proteins (Conti et a l, 1998; Jacobs and Harrison, 1998).1.10.1.4 Activation and Degradation of the IkBs
In response to diverse stimuli including proinflammatory cytokines, mitogens and several viral proteins, active NFkB translocates to the nucleus as a result of the complete proteolytic degradation of the IkB proteins or partial degradation of the pi 05 and pi GO precursors (Israel, 2000). Located within the N- terminal regulatory region of the IkB proteins are two essentially invariant serine residues. Homologous serines are also present in IkBc (Baeuerle and Baltimore, 1996) and in the Drosophila IkB homologue. Cactus (Reach et a l, 1996). These residues lie within a DSGv)/XS motif (Figure 1-9) and are phosphorylated by a large multi-kinase complex upon cell activation (Brown et a l, 1995; Chen et a l, 1996; Karin and Delhase, 2000). In IxBa, serine residues 32 and 36 are phosphorylated, followed by polyubiquitinated of lysine residues 21 and 22 by a specific E3 ubiquitm ligase complex (DiDonato et a l,
1996; Scherer et a l, 1995; Spencer et a l, 1999; Traenckner et a l, 1995; Winston et a l, 1999; Yaron et al.,
1998). This triggers the rapid degradation of the protein by the 26S proteasome (Alkalay et a l, 1995; DiDonato et a l, 1995). All members of the IkB femily are degraded/processed by a similar mechanism. For example, IkBP is phosphcxylated at serine residues 19 and 23 upon stimulation with extracellular agents, although with slower kinetics than those of IxBa phosphorylation (DiDonato et a l, 1996). The defining characteristic oflxB a is its ability to regulate rapid, but transient induction of NFkB activity.
k B s ite s are p resen t w ith in th e Ik B q p ro m o ter and f o llo w in g N F k B a c tiv a tio n , th e tran scrip tion o f I k B q
i t s e lf is u p reg u la ted , a llo w in g Ik B œ to sh u t o f f th e sig n a l (B r o w n et al., 1 9 9 3 ; C h ia o et a l , 1994; d e M artin et a i, 1993; L e B a il et a i, 1993; S c o tt et a i, 1 9 9 3 ; Sun et a i, 1 9 9 3 ). T h is a llo w s I k B q to b e in v o lv e d in both th e a c tiv a tio n an d in a ctiv a tio n o f g e n e tran scrip tion th rou gh its a ss o c ia tio n w ith N F k B . R ecen t stu d ies
s u g g e s t a m o d e l in w h ic h a g e n ts that in d u c e p ersisten t N F k B a c tiv ity in d u c e b oth I k B q an d Ik B P
d eg ra d a tio n . T h e n e w ly s y n th e s is e d Ik B P is u n p h o sp h o ry la ted an d a c ts a s a ch a p e r o n e o f N F k B , b lo c k in g
th e in h ib ito ry e ffe c t o f iK B a a n d m a in ta in in g N F k B a c tiv ity e v e n after I k B q r e s y n th e s is (S u y a n g et a i, 1 9 9 6 ; T h o m p so n et a i, 1 9 9 5 ). T h is is su p p orted b y stu d ie s in I k B o d e fic ie n t m ic e , w h ic h s u g g e s t that iK B a p la y s a p r ed o m in a n t r o le in th e p o s tin d u ctio n a l r ep ressio n o f N F k B a c tiv ity ( B e g et a i, 1995a; K le m en t £?/a /., 1 9 9 6 ). IkBœ 26 13 IkB P I k B c 151
Cactus
68Consensus
L D D R H D G L D M K D E E Y E Q A D E W C D G L G L G P D A A A P E E S Q Y D G I E P N E T S D G D S G Y % SF igu re 1-9: A lign m en t o f the N -term inal R egu latory R egion s o f th e IkB ’s and C actus. Figure redrawn from (Karin and Delhase, 2000). The critical serine residues (S) becom e phosphorylated upon cell stimulation, allow ing
recognition by the subunit o f the E3 ubiquitin ligase com plex. Ubiquitination and proteasomal degradation then