SOCS-3 Inhibits E2F/DP-1 Transcriptional Activity and Cell Cycle Progression via Interaction with DP-1

Abstract

Recent studies using SOCS family knock-out mice have suggested that SOCS proteins have multiple biological functions in addition to their role as negative regulators of JAK-STAT signaling. To explore these other functions of this family of proteins, we used yeast two-hybrid screening to find proteins interacting with human SOCS-3. We identified the transcriptional factor DP-1 as a SOCS-3-interacting protein involved in regulation of the cell cycle. Immunoprecipitation-Western blot assay showed that this interaction between these endogenous proteins occurred in cells both in vitro and in vivo. SOCS-3 interacted with the C-terminal region of DP-1, and amino acids 156 -172 of SOCS-3 were required for this interaction. Confocal microscopy revealed that SOCS-3 and DP-1 were primarily colocalized in the cytoplasm. SOCS-3 inhibited E2F/DP-1 transcriptional activity under the cyclin-E promoter and actually inhibited cell cycle progression and cell growth under E2F/DP-1 control. In contrast, DP-1 almost completely eliminated the inhibitory action of SOCS-3 on LIF-stimulated STAT-3 transcriptional activity in JAK-STAT signaling. Interestingly, the alternative regulatory action of SOCS-3 and DP-1 was dramatically eliminated by each siRNA. Taken together, these findings demonstrate that SOCS-3 acts as a negative regulator of the cell cycle progression under E2F/DP-1 control by interfering with heterodimer formation between DP-1 and E2F and also that DP-1 plays an important role in controlling JAK-STAT signaling.Members of the family of suppressor of cytokine signaling (SOCS), 2 designated SOCS-1 to SOCS-7 and CIS, are induced by stimulation via several kinds of cytokines and growth factors (1-3). These proteins regulate JAK-STAT signaling in a classical negative feedback loop of the signaling cascade (4, 5). SOCS family proteins also act as an important regulator of cell differentiation, as evidenced by the following findings: SOCS-1 suppresses muscle differentiation (6); SOCS-2 regulates neuronal differentiation (7); SOCS-3 induces myoblast differentiation (8); and SOCS-3 and SOCS-5 are involved in T helper cell differentiation (9, 10). In addition, these proteins are thought to strongly contribute to the development and progression of several kinds of tumors such as hepatocellular carcinoma (11-13), chronic myeloid leukemia (14), ovarian and breast carcinoma (15), and so on (16 -21). These observations suggested to us that SOCS family proteins might exhibit multipotential functions as regulators of cell differentiation and tumor cell growth besides being a negative regulator of JAK-STAT signaling. To explore this possibility, we considered that identification of SOCS-interacting proteins would be an extraordinarily good strategy. Thus, using the yeast two-hybrid screening system, we sought to identify presently proteins interacting with human SOCS-3. As a result, we found DP-1, a transcriptional factor for cell cycle regulation, to be such a SOCS-3-interacting protein.DP-1 was first identified as a partner protein of E2F-...

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