Eyal Kalie

The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities.

Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNα2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC50 values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNα2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.

Binding and activity of all human alpha interferon subtypes

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5-5 μM and 0.4-5 nM respectively (except for IFN-alpha1 - 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC50 values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.

An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

All α-interferons (IFNα) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFNα2. Here, we constructed a phage display library by randomizing three positions on IFNα2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S (YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFNα2, and 3-fold tighter compared with IFNβ. Binding of YNS to IFNAR2 was comparable with wild-type IFNα2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFNα2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFNα2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFNα2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency.

MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation

The ERK5 signaling cascade acts through sequential activation of MEKK2/3, MEK5 and ERK5 and transmits signals to a variety of stress and mitogenic related targets. In this study we examined the subcellular localization of the components of the ERK5 cascade and found that in resting, as well as in EGF-stimulated HeLa and Rat-1 cells, endogenous ERK5 is localized mainly in the nucleus. This location is different from the previously described location of exogenous ERK5, in the cytosol of resting cells, which is confirmed in this study. The reason for the different localization could be a saturation of anchoring moieties by the endogenous ERK5. Indeed, in situ detergent extraction analysis using Nonidet P-40, revealed that ERK5 is bound to detergent resistant moieties in the nucleus, while the exogenous protein fails to interact with those anchors. The upstream activator MEK5 is also localized in the nucleus both before and after EGF stimulation and is resistant to NP-40 extraction in resting cells. ERK5 remains bound to these nuclear moieties even after stimulation, while MEK5 is detached from the anchors but remains localized in the nucleus. Unlike ERK5 and MEK5, their upstream activator MEKK2 is localized mainly in the cytosol of resting cells, and translocates into the nucleus upon EGF stimulation, allowing transmission of signals to the nuclear MEK5. The nuclear localization of MEK5 and ERK5 is different from that of ERK1/2 and MEK1/2 in resting cells, indicating that each MAPK cascade uses distinct mechanisms to transmit extracellular signals to their nuclear targets.

Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.