Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecular stress sensor and response mediator implicated in multiple cellular functions in health and diseases. Despite its importance and intrinsic involvement in pivotal molecular and cellular processes, including DNA repair, transcription regulation, chromatin organization, and cell death, the regulatory mechanisms of PARP1 are poorly understood. In this study, we show that SMURF2, a HECT-type E3 ubiquitin ligase and suggested tumor suppressor, physically interacts with PARP1 in different cellular settings, directly ubiquitinates it in vitro and stimulates its PARylation activity in cells, the phenomenon that required SMURF2 E3 ubiquitin ligase function. Intriguingly, in the cellular environment SMURF2 was found to regulate the dynamic exchange of ubiquitin moieties on PARP1, mostly decreasing its monoubiquitination. Through the set of systematic mass spectrometry analyses conducted on SMURF2-modified cells, we identified on PARP1 18 lysine residues (out of 126 present in PARP1) as sites which ubiquitination was considerably affected by SMURF2. Subsequent site-directed mutagenesis coupled with in cellula ubiquitination and PARylation assays unveiled K222 as a critical site enabling a cross talk between SMURF2-modulated monoubiquitination of PARP1 and its activity, and pointed to K498, S507, and a KTR triad (K498/K521/K524) as the main auto-PARylation sites affected by SMURF2. The results also uncovered that SMURF2 controls PARP1 interactome, influencing its functions and expression in a context-dependent manner. Taken together, these findings suggest that SMURF2-mediated ubiquitin signaling plays an essential role in PARP1 regulation, beyond the regulation of its protein expression.

KAP1 is an essential nuclear factor acting as a scaffold for protein complexes repressing transcription. KAP1 plays fundamental role in normal and cancer cell biology, affecting cell proliferation, DNA damage response, genome integrity maintenance, migration and invasion, as well as anti-viral and immune response. Despite the foregoing, the mechanisms regulating KAP1 cellular abundance are poorly understood. In this study, we identified the E3 ubiquitin ligase SMURF2 as an important regulator of KAP1. We show that SMURF2 directly interacts with KAP1 and ubiquitinates it in vitro and in the cellular environment in a catalytically-dependent manner. Interestingly, while in the examined untransformed cells, SMURF2 mostly exerted a negative impact on KAP1 expression, a phenomenon that was also monitored in certain Smurf2-ablated mouse tissues, in tumor cells SMURF2 stabilized KAP1. This stabilization relied on the unaltered E3 ubiquitin ligase function of SMURF2. Further investigations showed that SMURF2 regulates KAP1 post-translationally, interfering with its proteasomal degradation. The conducted immunohistochemical studies showed that the reciprocal relationship between the expression of SMURF2 and KAP1 also exists in human normal and breast cancer tissues and suggested that this relationship may be disrupted by the carcinogenic process. Finally, through stratifying KAP1 interactome in cells expressing either SMURF2 wild-type or its E3 ligase-dead form, we demonstrate that SMURF2 has a profound impact on KAP1 protein–protein interactions and the associated functions, adding an additional layer in the SMURF2-mediated regulation of KAP1. Cumulatively, these findings uncover SMURF2 as a novel regulator of KAP1, governing its protein expression, interactions, and functions.

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