Tyrosine phosphorylation is a fundamental step in many signal transduction cascades required to mediate cellular processes. The Src homology 2 (SH2) domain forms the largest family of phosphotyrosine recognition domains, with a total of 120 different SH2 domains found in 110 distinct proteins in the human genome. The binding specificities between different SH2 domains and the sequence surrounding the phosphotyrosine motif in individual proteins guarantee the accurate recognition of the target protein and tightly regulated signal transmission. The SH2 domain is a small protein module consisting of three β-strands flanked by two α-helixes. These secondary structural elements and the loops that connect them form two pockets; one is the highly conserved phosphotyrosine binding pocket, and the second is a hydrophobic pocket that typically accommodates hydrophobic residues in the target protein located in the “+3” position following the phosphotyrosine.
Members of the Suppressors of Cytokine Signaling (SOCS) protein family are the primary negative regulators of Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling. Eight members have been identified, including CIS and SOCS1-7, which are defined by a central SH2 domain and a C-terminal SOCS box motif. The SOCS-SH2 domain conforms to the canonical SH2 domain structural fold and is distinguished by an additional α-helix N-terminal to the domain, designated the extended SH2 subdomain (ESS). Although some phosphotyrosine motif binding preferences have been defined for individual SOCS-SH2 domains, the full suite of protein targets and preferred binding sequences for each SOCS-SH2 domain remain to be elucidated. The SOCS proteins themselves have clearly defined biological roles in regulating signaling cascades activated by different cytokines and growth factors. The SOCS-SH2 domain is central to their function and in all instances tested, loss of SH2 binding to phosphotyrosine causes loss of SOCS activity. The SOCS box, a conserved 40-residue motif at the C-terminus of the SH2 domain, forms two alpha-helices and is constitutively associated with the adaptor proteins Elongin B and Elongin C, which function to both stabilize the SH2 domain and recruit an E3 ubiquitin ligase complex consisting of Cullin5 and RING-box protein (Rbx)2. This recruitment allows the ubiquitination and proteasomal degradation of targets bound to the SOCS-SH2 domains and classes the SOCS proteins as Cullin-RING E3 ligases (CRLs).
SOCS2 is the predominant negative regulator of growth hormone (GH) and prolactin signaling. Consequently, SOCS2-deficient mice (Socs2 −/−) display gigantism, weighing significantly more than their wild-type counterparts at 6 weeks of age, due to enhanced GH signaling. SOCS2 regulation of GH signaling is thought to primarily occur via SOCS2-SH2 domain recognition of phosphotyrosine Tyr595 in the GH receptor (GHR). However, the paradoxical observation that mice expressing a Socs2 transgene also display gigantism, remains mechanistically unexplained. Several reports indicate an important role for SOCS2 in immune cells, with Socs2 −/− mice displaying hyper-responsiveness to microbial stimuli in dendritic cells (DCs) and skewed T helper type 2 (Th2) differentiation and impaired generation of inducible regulatory T cells (iTregs). In addition, Socs2 is upregulated by interferon-gamma (IFNγ) in DCs in human melanoma, and Socs2 −/− mice exhibit enhanced DC priming of Tcell responses to give improved antitumor immunity.
SOCS2 has been reported to inhibit both STAT activation and NF-κB signaling; regulating the nuclear translocation of the NF-κB p65 subunit in DCs, with primary Socs2 −/− hepatocytes showing increased phospho-p65 in the nucleus compared with wild-type cells. SOCS2 clearly has many diverse roles in biological processes that are important for cancer, autoimmunity, and other inflammatory diseases. Understanding the function and recognition specificity of the SOCS2-SH2 domain is critical to understanding how SOCS2 regulates these different signaling cascades.
Here, we have discovered an exosite on the SOCS2-SH2 domain that, when occupied by a peptide (F3), reciprocally enhances the affinity of the SH2 domain for its canonical phosphorylated ligands. X-ray crystallography and NMR analysis revealed F3-bound as an α-helix on the opposite side to the phosphotyrosine binding pocket. F3 binding appeared to stabilize the ESS helix and phosphotyrosine binding loop, resulting in a reduced dissociation rate and increased SH2 affinity for its phosphorylated protein targets. Apart from the closely related CIS-SH2 domain, F3 binding was highly selective for SOCS2, with CIS displaying similar, although reduced, enhancement of affinity for phosphorylated ligands. This suggests the F3:exosite phenomenon might be applicable to other SH2 domains, with the cooperative increase in binding affinity implying an unappreciated degree of regulation. Expression of F3 peptide in A549 cells enhanced SOCS2-SH2 binding to the GHR and increased SOCS2 inhibition of GH signaling. Consequently, biophysical measurement of SH2-binding affinities might be substantially underestimated, particularly if both exosite and canonical sites are simultaneously occupied by proteins in the same complex.
In order to identify peptides that could bind to the surface of the SOCS2-SH2 domain, we used a highly degenerate phage-displayed library encoding random hexadecapeptides containing combinat
