Abstract
The serine/threonine protein kinase Akt1 plays an important role in mammalian cell growth, proliferation, migration, and angiogenesis and is activated by phosphorylation. To monitor threonine 308 phosphorylation in Akt1, we designed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide.
A phage display library of variant yeast Rad53p forkhead-associated 1 (FHA1) domain was screened by affinity selection for the phosphopeptide 301-KDGATMKpTFCGTPEY-315 yielding 12 binding clones. The strongest binders have equilibrium dissociation constants of 160-180 nanomolar and are phosphothreonine specific in binding. The specificity of an Akt1 pTBD was compared to commercially available polyclonal antibodies (pAbs) raised against the same phosphopeptide . Akt1-pTBD was either equal to or better than three pAbs in detecting the phosphopeptide Akt1 pT308 in ELISAs.
introduction
The serine/threonine protein kinase Akt1 is responsible for regulating a number of biochemical signaling pathways involved in cell proliferation and survival. It contains an N-terminal pleckstrin homology (PH) domain, a catalytic serine/threonine kinase domain and a C-terminal regulatory domain [1,2].
- Activation of Akt1 depends on the recruitment of the protein by its PH domain [3] to the inside of the plasma membrane, causing a conformational change [4] that allows PDK1 to target threonine 308 (T308) in the Akt1 catalytic domain phosphorylate and mTORC2 to phosphorylate serine 473 (S473) in the Akt1 regulatory domain [5,6].
- Once these two residues are phosphorylated, Akt1 is fully active and phosphorylates a number of intracellular proteins involved in cell survival, growth, proliferation, cell migration and angiogenesis [7].
- Given the crucial role Akt1 plays in the cell, biologists are very interested in understanding its involvement in cancer. Mass spectrometry and phosphospecific antibodies have been essential tools to pursue this question by monitoring the phosphorylation status and levels of Akt1 in cells and tissues [8,9].
- Such methods have shown a strong association between the hyperactivation of Akt1 by increased levels of phosphorylation in breast, prostate [10], ovarian [11] and pancreatic cancer [12]. In addition, studying the phosphorylation of specific residues within a protein can provide valuable information, as some diseases are characterized by the over -phosphorylation of just one or a few of these residues.
- For example, phosphorylation of T308 but not S473 has been characterized as a marker for lung cancer [13]. Therefore, antibodies that recognize specific phosphorylated residues as part of their epitopes serve as valuable diagnostic tools to discriminate between diseases caused by Akt1 deregulation.
- Unfortunately, mass spectrometry is not well suited to monitor protein phosphorylation at the cytological level, and antibodies are often poorly validated, not sequenced, and not amenable to protein engineering [14].
- To circumvent these limitations, current efforts have focused on generating modified protein scaffolds that recognize phosphoepitopes, such as the 10th fibronectin type III domain (10FnIII) [15], designed ankyrin repeat proteins (DARPins) [16], the Src homology 2 domain (SH2) [17], single-chain variable fragments (scFv) [18], antigen-binding fragments (Fab) [19] and the forkhead-associated (FHA) domain [ 20].
- Unlike other scaffolds and most antibodies, FHA domains are selective for phosphothreonine (pT)-containing targets due to a pocket on the domain that interacts with phosphate and the γ-methyl group of phosphothreonine (pT) [21,22]. Because of this unique property, a phage library containing randomized FHA1 variants at residues 82–84 in the β4-β5 loop and residues 133–139 in the β10-β11 loop was used to generate affinity reagents for a variety of targets [ 20, 21, 23].
- Here we describe the isolation and characterization of Akt1 phosphothreonine 308 (pT308) binding reagents. We show that these reagents are pT-dependent, bind with high affinity, and recognize the phosphopeptide with comparable or better specificity than commercially produced antibodies.
results and discussion
Directed evolution of the FHA1 domain resulted in variants recognizing an Akt1 phosphopeptide.
To generate recombinant affinity reagents that recognize Akt1-pT308, a peptide containing a phosphothreonine (pT) residue at position 308 in a 13-mer peptide (positions 302 to 314) with a C-terminal biotin was synthesized (Figure 1). A phage display FHA library containing 2 × 109 members [20] was affinity selected with the Akt1 pT308 peptide. After three rounds of affinity selection, 96 recovered clones were analyzed by ELISA and 12 were confirmed to bind the Akt1 pT308 peptide (Figure 2). None of the 12 binding clones showed any binding to the unphosphorylated form of the 302-314 Akt1 peptide.
DNA sequencing of 12 selected FHA domains showed that all were unique. A comparison of their coding sequences revealed that many of them shared amino acid sequences in the two regions randomized in the FHA domain framework (Figure 3A). The consensus motif in the β4-β5 and β10-β11 loops determined by a WebLogo plot [24,25] is (S/A)Y(Y/R) and (S/T)(P/A) x(R/I)(P/E)(S/D)(H/A) respectively (Figure 3B). We conclude from this findingthat the semi-conserved positions contribute to binding of the Akt1-pT308 peptide. Correlating with this observation, clones E12 and H11 closely resemble the consensus sequence and are the strongest binders, while clones E1 and B3 are more divergent and are the weakest binders. Among the residues most commonly shared by the binders is tyrosine at position 83, consistent with previous conclusions that position 83 is extremely important for the interactions of the FHA domain with its phosphopeptide targets [23,26,27].
Two of the affinity-selected FHA domains bind the Akt1 phosphopeptide with high affinity. To assess the binding strength of the affinity-selected FHA domain clones for the pT308 peptide, we performed competitive binding assays. The half-maximal inhibitory concentration (IC50) values for clones B3, E1, E12 and H11 were 100 μM, 1.95 μM, 45 nM and 90 nM, respectively. (Figure 4A). These relative values correlated well with the ELISA results: clone B3 was the weakest, clones E12 and H11 were the strongest binders, and clone E1 was an intermediate binder (Figure 4B). We then performed surface plasmon resonance (SPR) for two clones, E12 and H11, and determined equilibrium dissociation constants (KD) of 162 ± 12 nM and 178 ± 8 nM, respectively (Table 1). These clones are among the strongest binders that we have isolated [18].
An isolated FHA variant recognizes the Akt1 phosphopeptide with unique specificity.
Because of its unexpectedly high affinity for its phosphopeptide ligand, we decided to investigate clone E12 further. The specificity of this FHA variant was examined by testing the binding to peptides that replaced phosphothreonine (pT) in the phosphopeptide with phosphoserine (pS) or phosphotyrosine (pY). As can be seen in Figure 5, E12 only binds the peptide when position 308 is pT. On the other hand, three commercially produced polyclonal antibodies raised against the same or a similar Akt1 phosphopeptide differed in their specificity. While one antibody (pAb 1) showed a similar level of selectivity as clone E12, another (pAb 2) lacked specificity, and a third did not bind any of the peptides to determine which residues in the peptide ligand contributed to binding to clone E12 we tested a range of alanine-scanned phosphopeptides.
Seven biotinylated peptides, each position of which was individually replaced with alanine, were captured into Neutravidin-coated microtiter wells and probed separately with a pAb and clone E12. The pAb bound most of the alanine-scanned peptides as well or better than the wild-type sequence, with the exception of the alanine replacement at positions -3 and +2 in the phosphopeptide sequence. (Usually, the phosphorylated amino acid is defined as 0, and the N-terminal and C-terminal amino acids are numbered – and +, respectively.). However, the pAb did not bind to the unphosphorylated peptide or to a peptide with alanines flanking the central pT position. Conversely, the E12 clone showed very little or no binding to all test peptides.
Phosphothreonine Antibody (APC) |
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Hemoglobin (HB) Polyclonal Antibody (Rabbit), APC |
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Acetylcholinesterase (AchE) Polyclonal Antibody (Human), APC |
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Acetylcholinesterase (AchE) Polyclonal Antibody (Mouse), APC |
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Acetylcholinesterase (AchE) Polyclonal Antibody (Mouse), APC |
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In addition, it had a reduced ability to bind corresponding peptides from the Akt2 or Akt3 isoforms, whose sequences differ by only one or two amino acids at positions not tested by the alanine scan. This was surprising as previous observations show that FHA domains recognize only a few nearby residues in addition to the central pT. In this case, however, many of the residues flanking the central pT in the Akt1 phosphopeptide appear to directly or indirectly contribute to binding. Preliminary molecular dynamics studies of the peptide as well as the elucidated structure of activated Akt [28] suggest that hairpin loops form on either side of the pT residue, which could explain why so many of the neighboring residues are directly or indirectly important for the binding interaction.