lie at the beginning o f the kinase domain is shown in blue and occurs seven am ino acid residues upstream if
the first glycine found in the consensus m otif G -x-G -x-x-G -x-V . The boxed region shows the intermediate
region o f lRAK-1 which is located between the death domain and the kinase domain. A putative TRAF6
The kinase domain contains 11 conserved subdomains, ammg which are at least 15 conserved residues that are responsible for creating a catalytic core structure that is required to bind ATP when complexed with the divalent catim Mg^^. The kinase domain also binds the protein or peptide substrate in the correct orientation to catalyse transfer of the y-phosphate of ATP to the acceptor hydroxyl residue on serine, threonine w tyrosine residues of the substrate. Different members of this femily of proteins possess different substrate binding preferences and regulation modes, but the basic mechanism of y-phosphate transfer from the phosphate donor (ATP) to the hydroxyl group of the substrate (serine, threonine, tyrosine) is highly conserved (Hanks mid Hunter, 1995).
Based on structural predictions, IRAK I is made up of three distinct regions. The ultimate aim of this work was to define which regiwis of IRAK I might be important for particular aspects of IL-IRI signal transduction. In order to gen^ate the necessary constructs to study the functions of the different regions of IRAK-1, an initial analysis of the primary amino acid sequence was undertaken. As IRAK I contains a well-defined central kinase region, a convenimt sub-division of the IRAK-1 molecule was made based on the structurally distinct N-terminal, central (kinase) and C-terminal regicms. To define the amino acid residues located at the beginning and the end of the central kinase domain, the common conserved structural motife and the known 3-dimensional structures of members of the protein kinase superfemily were considered together with an analysis of the intron/exon arrangement of IRAK-1.
Hanks and HuntCT in 1995 aligned and analysed the amino acid sequences of 60 different kinase domains, identifying the amino acids that are responsible for their catalytic activity and defining 12 conserved kinase sub-domains (Hanks and HuntCT, 1995). This information was used to help with analysis o f the IRAK-1 protein. Due to the conservation of the kinase domains, it is likely that members of this superfemily fold into topologically similar three dimensional core structures. The structure is thought to consist of two lobes, a smaller lobe that is responsible for binding and orientating the nucleotide, (subdomains I-FV) and a larger carboxy-terminal lobe, (subdomains VIA-XI), which is considered to bind the substrate and initiate phospho-transfer. Subdomain V is thought to span the two lobes and a deep clefi: is formed wTiich is the catalytic site of the enzyme.
Amino acid sequence comparisons suggest that the IRAK-1 protein conforms to a similar basic structure. Hanks and Hunter highlighted the presence of highly conserved residues found among the different femily members using the type a cAMP-dependent protein kinase catalytic subunit (PKA-Ca) as a reference. Residues that are conserved in the IRAK-1 protein are indicated in red (mi figure 3-2. This structural conservation suggests that IRAK I will potaitially have a similar mode of action to PKA-Ca in terms of kinase catalytic activity.
The beginning of the kinase domain of IRAK-1 was defined according to the position of a conserved consensus motif G-x-G-x-x-G-x-V, which is found in subdomain I of protein kinases (underlined on figure 3-2). The amino-terminal boundary of the kinase domain occurs seven amino acid residues upstream of the first glycine residue found in the consensus motif (Hanks and Hunta", 1995). This is usually a hydrophobic amino acid and in the case of IRAK I is a phaiylalanine residue located at amino acid position 212 (highlighted in blue on figure 3-2). The first glycine residue of the consensus motif is located at position 219. It is possible to predict that IRAK I functions as a kinase by comparing key residues which are known to perform fimctimal roles within PKA-Ca. In PKA-Ca, the consensus motif referred to above consists of the amino acid residues G-T-G-S-F-G-R-V (Hanks and Hunter, 1995), Wiile in IRAK I the corresponding sequence is G-E-G-G-F-G-C-V (see Figure 3-2). The backbone amides (S-F-G) of PKA-Ca form hydrogen bonds with ATP ^-phosphate oxygens, while the valine residue, along with a leucine residue that directly precedes the consensus moti^ together contribute to a hydrophobic pocket that encloses the adenine ring of ATP (Hanks and Hunter, 1995). As can be seen in figure 3-2, IRAK I contains not only the conserved glycine and valine residues present within PKA-Ca, but also the phenyalanine residue. Additicmally, an isoleucine residue in IRAK I precedes the consensus motif, which shows homology to the leucine residue present at this position in PKA-Ca, suggesting that the two proteins function in a similar manner. Overall, subdomain I of IRAK-1 is likely to be responsible for anchoring the non-transferable phosphates (a, P) of ATP.
By id e n tif^ g conserved amino acids Wiich are present in both PKA-Ca and IRAK-1, it was possible to predict that IRAK I functions as a kinase. Lysine 239 and glutamic acid 259 in IRAK-1 are likely to form a salt bridge which stabilises interactions between the lysine residue and the a and P phosphates of ATP. This is important for anchoring and orientating ATP to produce maximal enzyme activity.
Aspartic acid 340 and asparagine 345 in subdomain VIB of IRAK-1 lie within the consensus motif H-R-D- L-K-x-x-N, also termed the catalytic loop of the enzyme. The aspartic acid residue within this motif is the catalytic base responsible for accepting protons from the hydroxyl group of the substrate (serine/threonine) during phosphotransfer. To confirm a similar role of D340 in IRAK-1, previous work in our laboratory used site-directed mutagenesis to replace this aspartic acid residue with an asparagine residue (D340N). Assessment of the catalytic activity of this construct in insect cells, confirmed that, unlike the wild-type enzyme, the mutant IRAK-1 was unable to autophosphorylate (Maschera et a l, 1999a). Asparagine 345 in IRAK-1 is likely to play a role in stabilising the catalytic loop by chelating the secondary Mg^^ ion that bridges the a and y-phosphates of ATP and by forming a hydrogen bond with D340.
As can be seen in figure 3-2, the lysine residue located within the catalytic loop (K342) is also conserved in IRAK-1. Again, by analogy to PKA-Ca, this residue may help facilitate phosphotransfer by neutralising the negative charge of the y-phosphate during transfer. Located within subdomain VII of PKA-Ca is a motif (DFG) that appears to be highly conserved among protein kinases. This DFG triplet is also found in IRAK-
1, corresponding to D358-F359-G360. Aspartic acid 358 is likely to be responsible for chelating primary Mg^^ ions which bridge the p and y-phosphates of ATP, orientating the y-phosphate for transfer. In subdomain VIII, glutamic acid 394 is involved in stabilising the large lobe by forming an ion pair with the conserved arginine residue in subdomain XI.
It appears that subdomain VIII is responsible for substrate recognition and binding, as well as containing residues which become phosphorylated to activate the kinase. In PKA-Ca, maximum kinase activity requires phosphorylation of threonine 197 through autophosphorylation, whereas members of the Erk (MAP) kinase family require dual phosphoryation of threonine 183 and tyrosine 185 for activation. Interestingly, equivalent conserved residues are present in IRAK-1 (T383, T387 and Y390) and are highlighted in green on figure 3-2. It is possible that phosphorylation of these residues may allow IRAK-1 to become activated due to stabilisation of the subdomain VIII catalytic loop, permitting proper orientation of the substrate. This may occur due to phosphorylation of IRAK-1 by an upstream kinase or by autophosphorylation. In PKA-Ca, asparticic acid 220 within subdomain IX plays a role in stabilising the catalytic loop by forming hydrogen bonds with arginine 165 and tyrosine 164 that precede the loop.
S im ila r ly , th is g lu ta m ic a c id is c o n s e r v e d in I R A K -1 , b u t e q u iv a le n t a r g in in e a n d ty r o s in e r e s id u e s are n o t,
s u g g e s tin g that E 3 9 4 m a y b e r e s p o n s ib le fo r th e k e y in te ra c tio n s. A su m m a r y o f th e k e y c o n s e r v e d
r e s id u e s in IR A K -1 an d th eir p o s s ib le fu n c tio n s is s h o w n in ta b le 3 -1 .
A m in o A c id
R e s id u e
S u b d o m a in P o s s i b l e F u n c t io n
F223 I Form hydrogen bonds with ATP P-phosphate oxygens
G 224 I
1218 I Contribute to the hydrophobic pocket that encloses the adenine ring o f
V 226 I ATP
A 237 II
K 239 II Form a salt bridge which stabilises the interactions between K 239 and the
E259 III a - and P-phosphates o f ATP. Essential for producing m aximum enzym e
activity