Both the Suppressor of Cytokine Signaling 1 (SOCS-1) Kinase Inhibitory Region and SOCS-1 Mimetic Bind to JAK2 Autophosphorylation Site: Implications for the Development of a SOCS-1 Antagonist

Abstract

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-γ by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-γ-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, (53)DTHFRTFRSHSDYRRI(68) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001–1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr1007, pJAK2(1001–1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-γ-induced STAT1-α phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-α activation. Both Tkip and SOCS1-KIR inhibited IFN-γ activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001–1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001–1013) enhanced suboptimal IFN-γ activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-γ activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001–1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.

Hematopoietic cytokine receptor signaling

“…SOCS1, via its SH2 domain, binds to Y1007 in the activation loop of JAK2. Originally identified as negative regulators of IL-6 signaling (Starr et al, 1997), subsequent studies have revealed that some SOCS proteins, such as SOCS1 and SOCS3, possess a kinase inhibitory region that is believed to compete with substrates of JAKs (reviewed in Khwaja, 2006) and peptides that correspond to this region can function as SOCS1 antagonists (Waiboci et al, 2007).…”

A Suppressor of Cytokine Signaling 1 Antagonist Enhances Antigen-Presenting Capacity and Tumor Cell Antigen-Specific Cytotoxic T Lymphocyte Responses by Human Monocyte-Derived Dendritic Cells

“…Third, pJAK2(1001-1013) enhanced IFN-␥ activation of the luciferase reporter gene via the IFN-␥-activated sequence promoter element. Fourth, pJAK2(1001-1013) enhanced antigen-specific splenocyte proliferation (23). Subsequently, pJAK2(1001-1013) was reported to enhance antiviral immunity (24).…”

“…On the basis of these findings, Waiboci and colleagues developed a small peptide antagonist of SOCS1, pJAK2(1001-1013), that corresponds to the activation loop of JAK2. Research results demonstrated that the pJAK2(1001-1013) peptide can block SOCS1-induced inhibition of STAT3 phosphorylation in IL-6-treated prostate cancer cells and enhance antigen-specific splenocyte proliferation (23). Later, they reported that, in addition to a direct antiviral effect and synergism with IFN-␥, the pJAK2(1001-1013) peptide exhibits adjuvant effects on humoral and cellular immunity, as well as an enhancement of polyinosinic-poly(C) activation of Toll-like receptor 3 (TLR3) (24).…”

SOCS1 downregulation in dendritic cells promotes memory T-cell responses

“…First, binding of a cytokine to its receptor upregulates expression of SOCS1. SOCS1 then inhibits JAK tyrosine kinase activity by binding to the catalytic site of Janus kinase tyrosine kinase (JAK), and by binding to and recruiting the ubiquitin-transferase complex to target JAK for proteasomal degradation [20,21]. SOCS1 also controls STAT and, indirectly, TLR signaling [22].…”