Deletion of the SOCS box of suppressor of cytokine signaling 3 (SOCS3) in embryonic stem cells reveals SOCS box-dependent regulation of JAK but not STAT phosphorylation

Abstract

The mechanism by which Suppressor of Cytokine Signaling-3 (SOCS3) negatively regulates cytokine signaling has been widely investigated using over-expression studies in cell lines and is thought to involve interactions with both the gp130 receptor and JAK1. Here, we compare the endogenous JAK/STAT signaling pathway downstream of Leukemia Inhibitory Factor (LIF) signaling in wild type (WT) Embryonic Stem (ES) cells and in ES cells lacking either the entire Socs3 gene or bearing a truncated form of SOCS3 (SOCS3DSB) lacking the C-terminal SOCS box motif (SOCS3DSB/DSB). In SOCS3DSB/DSB cells phosphorylated JAK1 accumulated at much higher levels than in WT cells or even cells lacking SOCS3 (SOCS3-/-). In contrast enhanced activation of STAT3 and SHP2 was seen in SOCS3-/- cells. Size exclusion chromatography of cell extracts showed that in unstimulated cells, JAK1 was exclusively associated with receptors but following cytokine stimulation hyperphosphorylated JAK1 (pJAK1) appeared to dissociate from the receptor complex in a manner independent of SOCS3. In WT and SOCS3DSB/DSB cells SOCS3 was associated with pJAK1. The data suggest that dissociation of activated JAK1 from the receptor results in separate targeting of JAK1 for proteasomal degradation through a mechanism dependent on the SOCS3 SOCS box thus preventing further activation of STAT3.

A critical role for SHP2 in STAT5 activation and growth factor–mediated proliferation, survival, and differentiation of human CD34+ cells

“…42,43 Reduced total JAK2 levels with chronic SHP2 knockdown may reflect enhanced proteasomal degradation of JAK2 because of activation of counter-regulatory proteins, such as suppressors of cytokine signaling 1 and suppressors of cytokine signaling 3. 44,45 Flow cytometry-based measurement of P-STAT5 in CD34 ϩ -expressing cells confirmed increased STAT5 phosphorylation at day 3 but not at day 5 of culture, indicating that these timedependent changes did not simply reflect the increased numbers of differentiated cells over time.…”

Three Lysine Residues in the Common β Chain of the Interleukin-5 Receptor Are Required for Janus Kinase (JAK)-dependent Receptor Ubiquitination, Endocytosis, and Signaling

“…However, it is well documented that JAK1 and JAK2 associate with suppressors of cytokine signaling 3 (SOCS3) and SOCS1 (53,54), respectively, and it is well established that SOCS proteins are negative feedback inhibitors of cytokine receptor signaling (55). SOCS1/3 are reported to interact with JAK1/2 via a central Src homology 2 domain and then act as adaptors via their SOCS box for the cellular ubiquitination machinery involving the Cullin⅐Elongin B/C complex (53,54). In this regard, it is tempting to speculate that in addition to ubiquitinating JAK1/2, the SOCS1/3⅐Elongin B/C ubiquitin ligase complex might also contribute to ␤c ubiquitination.…”

The SOCS box—Adapting proteins for ubiquitination and proteasomal degradation

“…This and other evidence would suggest that the target of SOCS3-mediated ubiquitination is JAK rather than the gp130 receptor subunit. For instance, ES cells expressing endogenous levels of SOCS3 lacking the SOCS box express elevated levels of phosphorylated Jak1 in response to LIF (46). In contrast, a series of studies from Irandoust and coworkers demonstrated that SOCS3 ubiquitinates lysine residues on the G-CSF receptor cytoplasmic domain, facilitating its trafficking to the lysosome (47,48).…”