Necdin Promotes Ubiquitin-Dependent Degradation of PIAS1 SUMO E3 Ligase

abstract: ...Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation.

... a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1

... that regulate protein degradation and turnover via the ubiquitin-proteasome pathway. Both ubiquitination and sumoylation utilize E1, E2, and E3 enzymes for their covalent modifications of target proteins [40] . Although numerous ubiquitin E3 ligases are involved in the specific substrate recognition, sumoylation relies on only a small number of E3 ligases such as PIAS family [41] , Cbx4 (Chromobox homolog 4)/Pc2 (Polycomb 2) [42] , Nsmce2 (non-SMC element 2)/Mms21 [43] , [44] , and RanBP2 (Ran

... sumoylation of PIAS1 substrate proteins and promotes PIAS1 degradation in the ubiquitin-proteasome pathway. We also show that the amino (N)- and carboxyl (C)-terminal regions of PIAS1 affect necdin-dependent ubiquitination. The present results provide novel insights into the regulatory mechanism of PIAS SUMO E3 ligase family by necdin and other MAGE family proteins. Results MAGE proteins differentially interact with SUMO E3 ligases We analyzed the interactions of typical SUMO E3 ligases including Nsmce2, Cbx4 and PIAS1 with MAGE proteins including necdin, MAGEA1, MAGED1, MAGEF1, necdin-like 2 and MAGEL2 by co-immunoprecipitation assay. To verify the assay system, we used the RING-type protein (non-SMC element 1) which interacts with necdin-like 2 and MAGEF1 but not with necdin, MAGED1 and MAGEL2 [38] . We found that Nsmce1 interacted with MAGEA1, necdin-like 2 and MAGEF1, but not with necdin, MAGED1 or MAGEL2 ( Fig. 1A ). We then examined the interactions of the SUMO E3 ligases Nsmce2, Cbx4, and PIAS1 with these MAGE proteins ( Fig. 1BD ). The MAGE proteins except MAGEA1 failed to interact with Nsmce2 and Cbx4 ( Fig. 1B, C ). Intriguingly, PIAS1 interacted with necdin, MAGEA1, MAGED1, MAGEF1 and MAGEL2, but not with necdin-like 2 ( Fig. 1D ). These data indicate that PIAS1 is a common target of diversified mammalian MAGE proteins. 10.1371/journal.pone.0099503.g001 Figure 1 MAGE proteins differentially interact with Nsmce1, Nsmce2, Cbx4 and PIAS1. ( A D ) Interactions of MAGE proteins with Nsmce1, Nsmce2, Cbx4, and PIAS1. Expression vectors for FLAG-tagged necdin (ND, 2 g), MAGEA1 (A1, 1 g), MAGED1 (D1, 8 g), MAGEF1 (F1, 4 g), necdin-like 2 (NL, 1 g), and MAGEL2 (L2, 6 g) were co-transfected with expression vectors for Myc-tagged Nsmce1 (Myc-Nsmce1, 3 g)( A ), Nsmce2 (Myc-Nsmce2, 3 g)( B ), Cbx4 (Myc-Cbx4, 3 g)( C ), and PIAS1 (Myc-PIAS1, 6 g)( D ) into HEK293A cells. Proteins were immunoprecipitated (IP) with anti-FLAG antibody (FLAG) and immunoblotted (IB) with anti-Myc (Myc) (top

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Gene: NSMCE2 - non-SMC element 2, MMS21 homolog (S. cerevisiae) - (NCBI)

Protein : 2yu4

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