In this study, we discovered that SENP1 controls lymphoid development through regulation of SUMOylation status of STAT5. Our data revealed that inactivation of SENP1 results in severe defects in early T and B cell development. We further showed that SENP1 deficiency causes accumulation of SUMOylated STAT5, resulting in inhibition of STAT5 activation and subsequent signaling. Moreover, biochemical studies indicated that both acetylation and SUMOylation occur on the same lysine residue in STAT5. SUMOylation of this lysine in STAT5 blocks its acetylation. These results suggest a critical role for SENP1 in regulating STAT5 transcriptional activity. SUMOylation of STAT5 observed in the absence of SENP1 appears to be lymphocyte specific, since no SUMOylated STAT5 could be detected in SENP1−/− myeloid cells.
Protein function is tightly regulated by reversible posttranslational modifications to create an on and off state that is crucial for many biological processes. Many proteins are dynamically modified at multiple sites by different modifications. The interplay between phosphorylation and SUMOylation of neighboring sites has been shown to play an important role in regulating the transcriptional activity of several transcription factors. For example, heat-shock factors (HSFs), GATA-1 and myocyte enhancer factor 2 (MEF2), containing a SUMO consensus site and an adjacent proline-directed phosphorylation site (ΨKxExxSP), are regulated by phosphorylation-dependent SUMOylation (Gregoire et al., 2006
; Hietakangas et al., 2003
; Hietakangas et al., 2006
). The motif ΨKxExxSP couples sequential phosphorylation and SUMOylation and has been referred to as a “Phospho-SUMOyl switch” (Yang and Gregoire, 2006
). We have identified a previously undescribed motif in which two SUMOylation sites of STAT5A at lysines 696 and 700 are located in close proximity to tyrosine 694, whose phosphorylation is a prerequisite for STAT5 activation. This suggests a possible interplay between SUMOylation and phosphorylation in regulating STAT5 activity. Indeed, SUMOylation of STAT5 is phosphorylation-dependent, since STAT5 phosphorylation mutant Y694A abolished SUMOylation of STAT5. We also found that SENP1 deficiency causes increased STAT5 SUMOylation, correlating with diminished STAT5 phosphorylation and activity in lymphocytes, indicating that SUMOylation of STAT5 inhibits its phosphorylation and subsequent signaling.
Since lysine can be a target of different posttranslational modifications, SUMOylation can block alternative lysine-targeted modifications, such as ubiquitination, methylation or acetylation. It has been reported that transcriptional activity of several transcription factors, such as SP3, HIC1 and MEF2A can be regulated by interplay between SUMOylation and acetylation on the same lysine residue (Sapetschnig et al., 2002
; Shalizi et al., 2006
; Stankovic-Valentin et al., 2007
). In addition to tyrosine phosphorylation, acetylation of different STATs has been shown to play a critical role in regulating their activity (Kramer et al., 2009
; Shankaranarayanan et al., 2001
; Tang et al., 2007
; Yuan et al., 2005
). For example, STAT3 acetylation at lysine 685 is essential for its dimerization and transcriptional activity (Yuan et al., 2005
). Here, our data clearly show that STAT5A is acetylated at lysine 696, which is also a target for SUMOylation. Acetylation of STAT5A at lysine 696 is essential for STAT5A activation, since mutation of this lysine diminished the transcriptional activity of STAT5A. Consistent with our finding, a recent study has reported that STAT5B acetylation on lysine 701 (corresponding to lysine 696 on STAT5A) is essential for STAT5B dimerization and transcription (Ma et al., 2010
). Notably, our data provide direct evidence that SENP1 regulates the activation of STAT5.
Based on the findings reported here, we propose a model for the role of SENP1 in the regulation of STAT5 activation (). Upon activation, tyrosine-phosphorylated and acetylated STAT5 dimerizes, translocates to the nucleus, and activates transcription. We currently do not know which signal induces SUMOylation of STAT5. SENP1 protein, which is predominantly present in the nucleus (Gong et al., 2000
), is required for de-conjugating SUMOylated STAT5 before it returns to the cytoplasm to complete an activation-inactivation cycle. In the absence of SENP1, STAT5 is accumulated in the SUMOylation state, leading to inhibition of STAT5 phosphorylation and acetylation, and subsequent signaling.
A model for the role of SENP1 in the regulation of STAT5 activation
It is not clear why SENP1 deficiency selectively affected the lymphoid, but not the myeloid lineage. There are several possibilities. First, lymphoid and myeloid precursors utilize different cytokines during development. For example, IL-7R signaling is critical for early lymphoid, but not myeloid development (Mazzucchelli and Durum, 2007
). Second, lymphoid cells may have a SUMO-specific E3 ligase that catalyzes SUMOylation of STAT5. This E3 ligase may not be present in myeloid precursors so STAT5 is not SUMOylated, which obviates the requirement for SENP1 to remove SUMO.
PIAS (Protein Inhibitor of Activated STAT) proteins were initially identified as negative regulators of STAT signaling that inhibit the activity of STAT-transcription factors (Chung et al., 1997
; Liu et al., 1998
). It has been shown that PIAS proteins function as SUMO-specific E3 ligases, raising the possibility that STAT activity might be regulated by the SUMOylation pathway (Schmidt and Muller, 2003
). PIAS3 is known to bind to STAT5 and suppress STAT5-mediated transcription (Rycyzyn and Clevenger, 2002
), but the precise molecular mechanism how PIAS3 negatively regulates STAT5 transcriptional activity is unknown. Although we also found that SUMOylation of STAT5 is greatly enhanced by PIAS3 in an overexpression system, further studies will be required to define whether this SUMO-specific E3 ligase is involved in regulation of STAT5 activity in lymphocytes.
In conclusion, SENP1 is essential for early T and B lymphopoiesis. Our data clearly demonstrate that SENP1 controls STAT5 activity by regulating the SUMOylation status of STAT5. Our findings establish a specific role of SENP1 in the regulation of STAT5 activation at the early stages of T and B cells.