RESUMO
In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Ligação Proteica/genética , Proteína SUMO-1/metabolismo , HumanosRESUMO
Cell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter cells are severed. Here, we present a novel abscission mechanism. We identified chromokinesin KIF4A, which is adjacent to the midbody during cytokinesis, as being required for efficient abscission. KIF4A is regulated by post-translational modifications. We evaluated modification of KIF4A by the ubiquitin-like protein SUMO. We mapped lysine 460 in KIF4A as the SUMO acceptor site and employed CRISPR-Cas9-mediated genome editing to block SUMO conjugation of endogenous KIF4A. Failure to SUMOylate this site in KIF4A delayed cytokinesis. SUMOylation of KIF4A enhanced the affinity for the microtubule destabilizer stathmin 1 (STMN1). We here present a new level of abscission regulation through the dynamic interactions between KIF4A and STMN1 as controlled by SUMO modification of KIF4A.
Assuntos
Mitose , Estatmina , Citocinese/genética , Proteínas de Ligação a DNA , Células HeLa , Humanos , Cinesinas/genética , Proteínas Nucleares , Estatmina/genéticaRESUMO
In contrast to our extensive knowledge on ubiquitin polymer signaling, we are severely limited in our understanding of poly-SUMO signaling. We set out to identify substrates conjugated to SUMO polymers, using knockdown of the poly-SUMO2/3 protease SENP6. We identify over 180 SENP6 regulated proteins that represent highly interconnected functional groups of proteins including the constitutive centromere-associated network (CCAN), the CENP-A loading factors Mis18BP1 and Mis18A and DNA damage response factors. Our results indicate a striking protein group de-modification by SENP6. SENP6 deficient cells are severely compromised for proliferation, accumulate in G2/M and frequently form micronuclei. Accumulation of CENP-T, CENP-W and CENP-A to centromeres is impaired in the absence of SENP6. Surprisingly, the increase of SUMO chains does not lead to ubiquitin-dependent proteasomal degradation of the CCAN subunits. Our results indicate that SUMO polymers can act in a proteolysis-independent manner and consequently, have a more diverse signaling function than previously expected.
Assuntos
Centrômero/metabolismo , Cisteína Endopeptidases/metabolismo , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cisteína Endopeptidases/genética , Células HEK293 , Células HeLa , Humanos , Interferência de RNA , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis.
Assuntos
Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Mitose/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Ubiquitinas/metabolismo , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase/genética , Linhagem Celular Tumoral , Células HCT116 , Células HeLa , Humanos , Cinesinas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinas/genéticaRESUMO
Post-translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Because little is known about crosstalk among different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk among different PTMs. In addition, various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin on inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, whereas the total protein level decreased immediately afterward. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.