Characterization of a Peptide Inhibitor of Janus Kinase 2 That Mimics Suppressor of Cytokine Signaling 1 Function

Abstract

Positive and negative regulation of cytokines such as IFN-γ are key to normal homeostatic function. Negative regulation of IFN-γ in cells occurs via proteins called suppressors of cytokine signaling (SOCS)1 and -3. SOCS-1 inhibits IFN-γ function by binding to the autophosphorylation site of the tyrosine kinase Janus kinase (JAK)2. We have developed a short 12-mer peptide, WLVFFVIFYFFR, that binds to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of IFN-γ receptor subunit IFNGR-1. The JAK2 tyrosine kinase inhibitor peptide (Tkip) did not bind to or inhibit tyrosine autophosphorylation of vascular endothelial growth factor receptor or phosphorylation of a substrate peptide by the protooncogene tyrosine kinase c-src. Tkip also inhibited epidermal growth factor receptor autophosphorylation, consistent with the fact that epidermal growth factor receptor is regulated by SOCS-1 and SOCS-3, similar to JAK2. Although Tkip binds to unphosphorylated JAK2 autophosphorylation site peptide, it binds significantly better to tyrosine-1007 phosphorylated JAK2 autophosphorylation site peptide. SOCS-1 only recognizes the JAK2 site in its phosphorylated state. Thus, Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not precisely the same way. Consistent with inhibition of JAK2, Tkip inhibited the ability of IFN-γ to induce an antiviral state as well as up-regulate MHC class I molecules on cells at a concentration of ∼10 μM. This is similar to the Kd of SOCS-3 for the erythropoietin receptor. These data represent a proof-of-concept demonstration of a peptide mimetic of SOCS-1 that regulates JAK2 tyrosine kinase function.

A SOCS-1 peptide mimetic inhibits both constitutive and IL-6 induced activation of STAT3 in prostate cancer cells

“…Intracellular delivery of Tkip via a lipophilic (N-terminal palmitylated) derivative inhibited IFNg antiviral activity and IFNg-mediated induction of MHC Class I (Flowers et al, 2004). It was also demonstrated that Tkip inhibits the autophosphorylation of EGFR by an unknown mechanism using in vitro kinase assays.…”

“…We previously developed a short 12-mer peptide mimetic of SOCS-1, WLVFFVIFYFFR (Tkip), that binds to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of gamma interferon (IFNg) receptor subunit IFNGR-1 (Flowers et al, 2004). Intracellular delivery of Tkip via a lipophilic (N-terminal palmitylated) derivative inhibited IFNg antiviral activity and IFNg-mediated induction of MHC Class I (Flowers et al, 2004).…”

“…Thus, Tkip specifically inhibited constitutive STAT3 phosphorylation in DU145 cells as well as IL-6-induced phosphorylation of STAT3 in LNCaP cells. Given that Tkip inhibits both JAK2 and EGFR tyrosine phosphorylations (Flowers et al, 2004), we next looked at the effect of Tkip on global tyrosine phosphorylation in DU145 and LNCaP cells treated with lipophilic Tkip in 20 h cultures. As can be seen in Figure 3a, untreated DU145 cells contained multiple phosphotyrosine proteins over a wide molecular weight range as determined by Western blot analysis of wholecell lysate gels with antiphosphotyrosine antibody (4G10).…”

“…DU145 cells were therefore treated with 1-8 mM lipophilic Tkip as shown in Figure 2a, and constitutive phosphorylation of 95 AKFEVNNPQVQRQAFNELIRVVHQLLPESSL 125 a The peptides used in this study were synthesized in our laboratory using conventional fluorenylmethyloxycarbonyl (Fmoc) chemistry as described previously (Szente et al, 1996;Flowers et al, 2004). A lipophilic group (palmitoyl-lysine) was added to the N-terminus of the synthetic peptides during peptide synthesis to enhance cellular uptake (Thiam et al, 1999) STAT3 was determined.…”

“…DU145 cells synchronized by serum starvation into G 0 /G 1 were stimulated to enter the cell cycle by serum addition in the presence or absence of 1-10 mM final concentrations of lipophilic Tkip (Flowers et al, 2004). Untreated cells and cells treated with lipophilic control peptide showed 30.6% each of cells in the S phase of the cell cycle at 24 h. Cells treated with 5 and 10 mM of Tkip showed 29.8 and 16.4%, respectively, of cells in the S phase of the cell cycle.…”