A clear picture has emerged in recent years showing that many members of the SOCS family have an important role in inhibition of the JAK/STAT pathway. The mechanisms by which CIS, SOCS1, and SOCS3 function to block cytokine signaling have been well documented (19
). All three have been shown to be induced rapidly and act in a feedback loop to inhibit JAK/STAT activity by binding the JAKs, the receptor, or both (6
). However, although SOCS2 can inhibit GH and PRL responses, it can also potentiate the signals induced by many cytokines. In this study, we found that, in contrast to other members of this family, SOCS2 appears to enhance cytokine signaling rather than suppress it.
To date, the mechanism of action of SOCS2 is unclear. One previous report suggested that SOCS2 may have a dual role (12
) since low concentrations of SOCS2 inhibited GH action and higher concentrations of SOCS2 enhanced GH signaling. Accordingly, both mice overexpressing SOCS2 and mice lacking SOCS2 displayed a gigantism phenotype (31
). This implies that SOCS2 can have a positive and negative role in GH signaling. It is therefore tempting to speculate that in other systems, SOCS2 acts as an accelerator rather than an inhibitor of cytokine signaling. Moreover, SOCS2-null mice show improved responses to growth hormone, indicating an enhanced effect on cytokine responses. However, higher levels of SOCS2 also block GH responses, suggesting that antagonism of other SOCS may occur in these cells. Our observations support the theory that expression of SOCS2 can enhance cytokine responses, most likely by driving degradation of other SOCS proteins.
We have demonstrated that both SOCS2 and SOCS3 are cytokine-induced genes in human PBMCs but that the proteins appear with different kinetics. SOCS3 protein was detected rapidly (30 min following stimulation), while SOCS2 did not appear until later. We also provide evidence that while SOCS3 inhibited both IL-2- and IL-3-induced tyrosine phosphorylation and proliferation, SOCS2 was able to enhance signaling. Since SOCS3 protein expression was maintained in the presence of proteasome inhibitors and mRNA levels of SOCS3 were not reduced by SOCS2, it was evident that SOCS2 at least partially enhanced signaling by reducing SOCS3 protein levels via a proteasome-dependent mechanism. These observations are further supported by the finding that IL-3-induced gene expression was also enhanced when SOCS2 was highly expressed. It is possible that SOCS2 blocks the expression of other SOCS family members, particularly since all SOCS proteins compete for binding to the same E3 ligases. Indeed, our preliminary data suggest that SOCS2 may also reduce SOCS1 expression (data not shown).
It is intriguing that cytokine-induced SOCS2 is expressed much later in peripheral blood T cells. This suggests that SOCS2 is not involved in the feedback loop that inhibits cytokine signaling but may limit this inhibition. Moreover, the finding that SOCS2 is still expressed 24 h after IL-2 treatment suggests that it may be important to potentiate the proliferation of these rapidly dividing cells, perhaps by keeping the expression of other SOCS at reduced levels. This is also true of IL-3-induced SOCS2 in Ba/F3 cells (J. A. Johnston, unpublished observations).
When expressed at high levels, SOCS2 has been demonstrated to antagonize the inhibitory effects of SOCS1 on PRL and GH signaling (5
) by an unknown mechanism. CIS levels are also lower in the presence of SOCS2, although the effects are not as marked as for SOCS3 (data not shown), and CIS is also induced later than SOCS1 or SOCS3 in response to cytokine stimulation (3
). Clearly, we have observed SOCS3 degradation in the presence of overexpressed SOCS2, but since both proteins are induced by many ligands and appear to be reciprocally regulated, the SOCS2-induced degradation of SOCS3 would presumably function under normal physiological conditions. This would provide a mechanism for eliminating SOCS3 and the other SOCS family members and thus resensitize cells for further cytokine-mediated responses.
The mechanism of action of SOCS2 remains unclear, although several theories have been suggested. One theory holds that high concentrations of SOCS2 may overcome the effect of endogenous SOCS3 on GH signaling (8
). SOCS3, unlike SOCS2, contains a kinase-inhibitory region at its N terminus which is thought to directly inhibit JAK activity (36
). It has been suggested that SOCS2 may compete with SOCS3 for binding to the GH receptor, and the lack of a kinase-inhibitory region on SOCS2 may result in the continuation of JAK activity and enhanced signaling (12
). However, recently, Greenhalgh et al. reported that SOCS2 binds to the GH receptor at Y487 and Y595, which are not classic immunoreceptor tyrosine-based inhibitory motifs, suggesting that SOCS3 will not compete to bind these sites (13
). Furthermore, the SH2 domains of SOCS2 and SOCS3 differ significantly and are therefore likely to interact with different phosphotyrosine sequences on different target molecules.
Since our data imply that in the presence of SOCS2, SOCS3 protein expression is downregulated, a more likely theory is that SOCS2 may compete with SOCS3 for binding to the elongin B/C complex. This interaction could occur via the BC box region of the SOCS box. Other SOCS box-containing proteins, including VHL (21
), Muf1 (24
), elongin A (1
), and SOCS1 (15
), are thought to be stabilized by this interaction. It has also been shown that tyrosine phosphorylation in the SOCS box of SOCS3 (14
) and serine/threonine phosphorylation of SOCS1 (2
) disrupt elongin binding. Therefore, SOCS2 may bind to or compete with SOCS3 for elongin B/C, resulting in reduced SOCS3 protein stability.
Another possibility is that SOCS2 may form part of an E3 ligase complex similar to that of VHL. The C-terminal domain of the VHL protein is homologous to the SOCS box and interacts with the elongin B/C complex which in turn binds the Cullin family member Cul2 and RING finger protein Rbx1 to form an E3 ligase complex (25
). Under normoxic conditions, hypoxia-inducible factor 1α binds to the VHL E3 ligase complex, resulting in ubiquitination and proteasomal degradation (20
). SOCS1 has been proposed to form part of an E3 ligase complex containing Cul5 and Rbx1 (24
) to target associated proteins for degradation. More recently, it has been suggested that SOCS1 lacks a Cul5 binding site within the SOCS box (26
); however, it is plausible that other Cullin proteins may interact with SOCS1.
As shown in Fig. , SOCS2 and SOCS3 can associate both in vitro and in vivo, although the precise nature of this interaction remains unknown. However, this study brings us a step closer to understanding when the interaction between SOCS2 and SOCS3 can occur. Our data imply that both proteins associate upon cytokine stimulation when proteasome activity is blocked. It is yet to be determined whether phosphorylation plays a role in regulating the SOCS2-SOCS3 association. Since we have observed association of these proteins, it is plausible to suggest that SOCS2 may act as a linker which brings an E3 ligase complex into close proximity with SOCS3. This may be a potential mechanism by which SOCS2 could result in the loss of SOCS3 protein.
SOCS1 has been reported to regulate the half-life of VAV (4
) and the insulin receptor substrates IRS1 and IRS2 (35
). Also, SOCS1 inhibits the kinase activity of JAK2 (41
) and the TEL-JAK2 oncogene (9
) in a phosphorylation-dependent manner by inducing SOCS box-dependent proteasomal degradation. SOCS1 therefore targets these substrates to the proteasome for degradation. Also, SOCS1 and SOCS3 promoted polyubiquitination and degradation of focal adhesion kinase in a SOCS box-dependent manner which inhibited focal adhesion kinase-dependent signaling events (30
). Likewise, the SOCS box has been implicated in the inhibition of granulocyte colony-stimulating factor signaling, suggesting a role for proteasomal degradation mediated via SOCS1 and SOCS3 in downregulating granulocyte colony-stimulating factor responses (42
). This evidence suggests that the SOCS box is involved in the proteasomal targeting of specific substrates, including perhaps other SOCS. Therefore, SOCS2 may act as part of an E3 ligase and target SOCS3, and perhaps other SOCS, for ubiquitination and degradation via the 26S proteasome.
Despite these observations, questions remain concerning how these findings relate to immune homeostasis and disease. SOCS3 deficiency leading to sustaining IL-6-induced STAT3 activation is thought to contribute to inflammatory diseases such as rheumatoid arthritis, Crohn's disease, and inflammatory bowel disease (38
). In comparison, sustained SOCS3 expression in T cells skews differentiation towards a Th2 response which may lead to Th2-related allergy (37
). It will be interesting to establish if altered SOCS3 expression in these instances is due to defects in the ability of SOCS2 to regulate SOCS3 expression.