The use of a random mutation library combined with a functional assay system is a powerful strategy for structural-functional analysis (32
). We used this approach combined with a yeast two-hybrid assay in order to identify the residues of TCL1 required for Akt binding. Enforced dNTP-driven PCR amplification was utilized to generate a random TCL1 library as described previously (15
). Since TCL1 cDNA consists of only 342 nucleotides, the approximately 3% substitution frequency resulted on the average in nine nucleotide (and one to three amino acid) substitutions per single TCL1 molecule, making further analysis by library screening feasible.
Although the yeast two-hybrid system was originally developed to identify protein-protein interaction (13
), our results indicate that it can be used to screen for loss of interaction. This was made possible by the very high β-Gal activity induced by the wild-type TCL1-Akt interaction in the system. It enabled mutant forms of TCL1 with very weak Akt interactions to grow on Trp-Leu-His-deficient selection plates and be identified using the β-Gal lifting assay. The screening was also relatively specific and possibly applicable to other molecules, since, of the five residues showing accumulation of mutations, two were found to be functionally relevant.
Analysis of the X-ray structure has revealed that TCL1 forms a closed symmetrical β-barrel structure, consisting of eight antiparallel β-strands. The barrel consists of two similar four-stranded β-meander motifs: motif 1, composed of strands A to D, and motif 2, composed of strands E to H. A long loop following strand D inverts the second motif by juxtaposing strand E to strand A. Each β-sheet of TCL1 contains two strands from each motif (23
). One sheet is composed of two pairs of the shorter strands (A-B and E-F), whereas the other is formed by the longer strands (C-D and G-H). The loop contains one helical turn (Pro-64-Gly-68) (Fig. ).
FIG. 6. Alignment of amino acid positions D16 and I74 within the topology of the eight-strand antiparallel β-sheets of TCL1. (A) The crystal structure of TCL1 (23) and the amino acid positions of D16 and I74 are shown. Amino acids D16 and I74 are located (more ...)
The results obtained in the screening of the random TCL1 mutation library and their functional verification demonstrated that amino acids D16 and, to a lesser extent, I74 mediate Akt binding. These residues are localized at very similar positions at the very beginning of the first β-strand of each of the two β-meander motifs of TCL1 (βA and βE, D16 and I74, respectively [Fig. ]). Moreover, they reside on the same flat surface, formed by βA and βE, of mature TCL1 (Fig. ). βA and βE are linked together in an antiparallel fashion by CO-to-NH hydrogen bonds (23
D16, which is hydrophilic and accessible to solvent, is likely to contribute to the stabilization of βA to βE through electrostatic interaction together with surrounding amino acids including a cluster of basic side chains of R17, E29, and Q77. Molecular modeling predicts that the substitution of glycine (G16) for aspartic acid (D16) changes the angle between amino acids 15 and 17 (PDR to PGR) from 88.45 to 71.62°, altering the alignment of βA to βE. In addition, the absence of a side chain towards the βE surface in glycine mutation (Fig. and , inset) might also have a role in destabilizing the interaction.
In contrast to D16, the role of I74 in Akt interaction seems less clear. Although in the yeast system, I74V mutation clearly disrupted Akt binding, its effects in mammalian cells and functional assays were less evident. I74 is located at the beginning of the βE-sheet, which is well conserved among the TCL1 family proteins. The residue appears to be poorly solvent accessible. It may participate in forming a long loop structure composed of amino acids 53 to 73 via a hydrophobic interaction with P64 and contribute to maintaining a local hydrophobic surface.
It is possible that Akt binds to TCL1 through the βA- to βE-sheets via two distinct sites: D16, conceivably together with the surrounding conserved amino acids, and the hydrophobic contact region P64/I74. Detailed molecular characterization of the interaction awaits the elucidation of the cocrystal structure of the Akt-TCL1 complex.
We have demonstrated elsewhere that TCL1 forms dimers as predicted by X-ray crystallographic studies (23
). The crystal study suggested that the βC-sheet of TCL1 acts as a dimerization domain (19
). Since the Akt-TCL1 interaction is mediated by the surface formed by βA and βE, it is logical that TCL1 dimerization can be mediated by the surface directly on the opposite side of the molecule and therefore targets the βC-sheet. Our data demonstrate that 36-38A and 36A/38Δ mutations, which presumably destroy the βC-sheet, eliminate the homodimerization of TCL1 but have no effect on Akt association. Based on this, the TCL1-TCL1 binding is conferred by interaction between the βC-sheets of two symmetrical TCL1 molecules.
How does the presence of TCL1 lead to enhanced Akt phosphorylation and kinase activation? It is well documented elsewhere that dimerization can lead to activation of surface receptor kinases (e.g., vascular endothelial growth factor receptor and platelet-derived growth factor receptor) and regulate their intracellular responses (3
). It is unclear whether intracellular nontransmembrane kinases can be activated through dimerization. It has been reported elsewhere that TEL-JAK2 fusion protein causes human leukemia due to oligomerization and constitutive kinase activation (27
). The Akt PH domain has been suggested previously to mediate dimerization (6
). Further, a conditionally activated Akt fused to the hormone binding domain of estrogen receptor was able to stimulate PHAS-1 phosphorylation (26
), suggesting that Akt dimerization may promote kinase activity.
PDK1 is required for phosphorylating Akt at Thr-305/308 (37
) and for activation of Akt, which is thought to be the prerequisite for subsequent Ser-473 phosphorylation (3
). However, recent studies using PDK1−/−
cells suggest that phosphorylation of Ser-473 may be independent from phosphorylation of Thr-308 (50
). Supporting this notion, Ser-473 of Akt was recently shown to be regulated by integrin-linked kinase (36
). Since wortmannin treatment can block the TCL1-dependent Akt activation, some degree of basal Akt activity seems to be required for the TCL1-dependent Akt kinase activation to occur (28
). This observation suggests not only that TCL1 functions within the intracellular phosphoinositide 3-kinase (wortmannin-sensitive) pathway but also that association of Akt with PIs and/or a certain preactivation of Akt, sensitive to wortmannin, may be prerequisite for the TCL1-induced Akt activation. Our observations also suggest that the effect of TCL1 on the phosphorylation status and activation of Akt seems to be additive to, and possibly at least partially independent of, the effects of PDK1. However, in our experimental system of in vitro kinase assays, we cannot completely exclude the potential contamination of immobilized Akt by PDK1 or other functionally equivalent kinases during the immobilization process from transfected cells.
The Akt PH domain can directly bind to phospholipids including PI-3,4-bisphosphate and PI-3,4,5-triphosphate, thereby facilitating the translocation of the protein to the plasma membrane (14
). As the molecular masses of phospholipids are relatively small (less than 5 kDa), it is feasible for the PH domain of Akt, which is composed of over 100 amino acids, to interact with PIs as well as TCL1.
As demonstrated by coimmunoprecipitation assays, our data show that TCL1 binds to Akt and facilitates the formation of Akt-TCL1 hetero-oligomers in vivo. Thereby, Akt molecules come into close physical proximity with each other. We have recently demonstrated that, in the presence of TCL1, Akt Ser-473 phosphorylation can result from transphosphorylation by other Akt molecules in the oligomers (28a
). In this scenario, PDK1 is required for triggering the Akt kinase activation and TCL1 further enhances Akt kinase activity. These data are in accordance with reports demonstrating the requirement of Akt kinase activity for Ser-473 phosphorylation in vitro (46
Therefore, although it is likely that unidentified kinases capable of Akt Ser-473 phosphorylation exist, at least under certain circumstances Ser-473 phosphorylation can occur through oligomerization-induced Akt-Akt transphosphorylation (Fig. ). Based on the findings of the present study, it seems likely that the TCL1-Akt interaction domain is required for the recruitment of individual Akt molecules into the oligomeric complexes and that the TCL1 dimerization domain is essential for bringing several Akt molecules into the same complex. Thus, both domains are needed for the full function of TCL1 as an Akt coactivator in vitro and in vivo (Fig. ).
The physiological expression of TCL1 is relatively abundant at early embryonic stages (34
) and during CD3-negative stages of T-cell development (49
), periods when external growth factor stimuli may be limited. TCL1 family proteins may function as a structural amplification loop in the phosphoinositide 3-kinase-Akt pathway, providing a survival advantage.